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.     

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.     

Shiga Toxin 2 Targets the Murine Renal Collecting Duct Epithelium

Hemolytic-uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli infection is a leading cause of pediatric acute renal failure. Bacterial toxins produced in the gut enter the circulation and cause a systemic toxemia and targeted cell damage. It had been previously shown that injection of Shiga toxin 2 (Stx2) and lipopolysaccharide (LPS) caused signs and symptoms of HUS in mice, but the mechanism leading to renal failure remained uncharacterized. The current study elucidated that murine cells of the glomerular filtration barrier were unresponsive to Stx2 because they lacked the receptor glycosphingolipid globotriaosylceramide (Gb₃) in vitro and in vivo. In contrast to the analogous human cells, Stx2 did not alter inflammatory kinase activity, cytokine release, or cell viability of the murine glomerular cells. However, murine renal cortical and medullary tubular cells expressed Gb₃ and responded to Stx2 by undergoing apoptosis. Stx2-induced loss of functioning collecting ducts in vivo caused production of increased dilute urine, resulted in dehydration, and contributed to renal failure. Stx2-mediated renal dysfunction was ameliorated by administration of the nonselective caspase inhibitor Q-VD-OPH in vivo. Stx2 therefore targets the murine collecting duct, and this Stx2-induced injury can be blocked by inhibitors of apoptosis in vivo.Shiga toxin-producing Escherichia coli (STEC) is the principal etiologic agent of diarrhea-associated hemolytic-uremic syndrome (HUS) (42, 60, 66). Renal disease is thought to be due to the combined action of Shiga toxins (Shiga toxin 1 [Stx1] and Stx2), the primary virulence factors of STEC, and bacterial lipopolysaccharide (LPS) on the renal glomeruli and tubules (6, 42, 60, 66). Of these, Stx2 is most frequently associated with the development of HUS (45). Shiga toxin enters susceptible cell types after binding to the cell surface receptor glycosphingolipid globotriaosylceramide (Gb₃) and specifically depurinates the 28S rRNA, thereby inhibiting protein synthesis (42, 60, 66). The damage initiates a ribotoxic stress response consisting of mitogen-activated protein (MAP) kinase activation, and this response can be associated with cytokine release and cell death (21, 22, 25-27, 61, 69, 73). This cell death is often caspase-dependent apoptosis (18, 61). Gb₃ is expressed by human glomerular endothelial cells, podocytes, and multiple tubular epithelial cell types, and damage markers for these cells can be detected in urine samples from HUS patients (10-12, 15, 49, 73). Shiga toxin binds to these cells in renal sections from HUS patients, and along with the typical fibrin-rich glomerular microangiopathy, biopsy sections demonstrate apoptosis of both glomerular and tubular cell types (9, 29, 31).Concomitant development of the most prominent features of HUS: anemia, thrombocytopenia, and renal failure, requires both Shiga toxin and LPS in the murine model (30, 33). Nevertheless, our previous work demonstrated that renal failure is mediated exclusively by Stx2 (33). While it is established that Gb₃ is the unique Shiga toxin receptor (46), the current literature regarding the mechanism by which Shiga toxin causes renal dysfunction in mice is inconsistent. Even though Gb₃ has been localized to some murine renal tubules and tubular damage has been observed (19, 23, 46, 53, 65, 68, 72, 74), the specific types of tubules affected have been incompletely characterized. Although multiple groups have been unable to locate the Shiga toxin receptor Gb₃ in glomeruli in murine renal sections (19, 53), one group has reported that murine glomerular podocytes possess Gb₃ and respond to Stx2 in vitro (40), and another group has reported that renal tubular capillaries express the Gb₃ receptor (46). Furthermore, murine glomerular abnormalities, including platelet and fibrin deposition, occur in some murine HUS models (28, 30, 33, 46, 59, 63). We demonstrate here that murine glomerular endothelial cells and podocytes are unresponsive to Stx2 because they do not produce the glycosphingolipid receptor Gb₃ in vitro or in vivo. Further, murine renal tubules, including collecting ducts, express Gb₃ and undergo Stx2-induced apoptosis, resulting in dysfunctional urine production and dehydration.     

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.     

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.     

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.     

Mobilization of CD34+ Progenitor Cells in Association with Decreased Proliferation in the Bone Marrow of Macaques after Administration of the Fms-Like Tyrosine Kinase 3 Ligand

Fms-like tyrosine kinase 3 ligand (FLT3-L) is critical for the differentiation and self-renewal of CD34⁺ progenitor cells in primates and has been used therapeutically to mobilize progenitor and dendritic cells in vivo. However, little is known regarding the expansion of progenitor cells outside of peripheral blood, particularly in bone marrow (BM), where progenitor cells primarily reside. Evaluation of FLT3-L-mediated cell mobilization during lentivirus infections, where the numbers of CD34⁺ progenitor cells are reduced, is limited. We enumerated frequencies and absolute numbers of CD34⁺ progenitor cells in blood and BM of naive and SIV- or SHIV-infected macaques during and after the administration of FLT3-L. Flow cytometric analyses revealed that, while CD34⁺ cells increased in the circulation, no expansion was observed in BM. Furthermore, in the BM intracellular Ki67, a marker of cell proliferation, was downregulated in CD34⁺ progenitor cells but was upregulated significantly in the bulk cell population. Although the exact mechanism(s) remains unclear, these data suggest that CD34⁺ cell mobilization in blood was the result of cellular emigration from BM and not the proliferation of CD34⁺ cells already in the periphery. It is possible that the decreased progenitor cell proliferation observed in BM is evidence of a negative regulatory mechanism preventing hyperproliferation and development of neoplastic cells.The cytokine receptor Fms-like tyrosine kinase 3 (FLT3) is expressed at high levels on both primitive and early lymphoid/myeloid CD34⁺ progenitor cells (3, 21). Interaction with its cognate ligand (FLT3-L), found in both soluble and membrane-bound isoforms, contributes to the regulation of self-renewal and differentiation potential of these cells (43, 44). However, dysregulation of FLT3/FLT3-L signaling can result in the development of various leukemias (1, 6, 22, 29), and increased serum levels of FLT3-L are often indicative of other hematologic and autoimmune abnormalities (17, 25, 39). Nonetheless, after both murine and human FLT3-L were cloned in the early 1990s (24), this hematopoietic cytokine was used effectively in vitro to expand and maintain CD34⁺ progenitor cells (26, 32, 33) and, in combination with other growth factors, was used to induce differentiation of myeloid lineage cells (4), dendritic cells (2, 15), natural killer (NK) cells (42), erythroid precursors (12), and even endothelial cells (41). In addition, FLT3-L was shown to specifically suppress apoptosis of CD34⁺ progenitor cells (27).Early in vivo studies in mice demonstrated that FLT3-L administration not only mobilized and expanded murine CD34⁺ progenitor cells but also promoted expansion of human CD34⁺ cells transferred into SCID mice (8, 9, 24). In nonhuman primates FLT3-L was used to expand dendritic cell subsets (7, 30, 35), to treat radiation-induced myelosuppression (13, 14, 19), and as an adjuvant for various vaccines (23, 40). Although CD34⁺ cells primarily reside in the bone marrow (BM), examination of mobilization of these cells in vivo in nonhuman primates has been limited and typically restricted to analyses of blood (5, 18, 28). CD34⁺ cell mobilization and hematopoiesis is of particular interest in macaque models of lentivirus infections because, during both HIV and SIV infections, BM damage and reduced hematopoiesis is evident early after infection and is associated with decreased numbers and clonogenic potential of CD34⁺ progenitors, despite low levels of infection and virus replication in these cells (10, 16, 20, 34, 36, 37). Therefore, in the present study we quantified and characterized mobilization of CD34⁺ progenitor cells in BM in relation to that observed in peripheral blood by examining BM aspirates taken at various times during and after FLT3-L administration to naive and SIV- or SHIV-infected macaques.     

Ail Binding to Fibronectin Facilitates Yersinia pestis Binding to Host Cells and Yop Delivery

Yersinia pestis, the causative agent of plague, evades host immune responses and rapidly causes disease. The Y. pestis adhesin Ail mediates host cell binding and is critical for Yop delivery. To identify the Ail receptor(s), Ail was purified following overexpression in Escherichia coli. Ail bound specifically to fibronectin, an extracellular matrix protein with the potential to act as a bridge between Ail and host cells. Ail expressed by E. coli also mediated binding to purified fibronectin, and Ail-mediated E. coli adhesion to host cells was dependent on fibronectin. Ail expressed by Y. pestis bound purified fibronectin, as did the Y. pestis adhesin plasminogen activator (Pla). However, a KIM5 Δail mutant had decreased binding to host cells, while a KIM5 Δpla mutant had no significant defect in adhesion. Furthermore, treatment with antifibronectin antibodies decreased Ail-mediated adhesion by KIM5 and the KIM5 Δpla mutant, indicating that the Ail-fibronectin interaction was important for cell binding. Finally, antifibronectin antibodies inhibited the KIM5-mediated cytotoxicity of host cells in an Ail-dependent fashion. These data indicate that Ail is a key adhesin that mediates binding to host cells through interaction with fibronectin on the surface of host cells, and this interaction is important for Yop delivery by Y. pestis.The three species of Yersinia pathogenic for humans, Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis, cause distinct diseases. Y. pseudotuberculosis and Y. enterocolitica typically cause acute gastroenteritis and mesenteric lymphadenitis. On the other hand, Y. pestis, the causative agent of the plague, is one of the most deadly human infectious diseases (8). Y. pestis is a close relative of Y. pseudotuberculosis, diverging only 1,500 to 20,000 years ago (1). To accommodate flea-borne transmission, Y. pestis has acquired two unique plasmids not harbored by enteropathogenic Yersinia species. All three pathogenic Yersinia species inject cytotoxic Yersinia outer proteins (Yops) into host cells via the Ysc type III secretion system (TTSS) to establish an infection (11). Host cell contact is essential for engagement of the TTSS and secretion of Yops (9, 54). Within the host cell, Yops effect actin rearrangements, inhibit phagocytosis, and block proinflammatory signals (4, 40, 42). Both Y. enterocolitica and Y. pseudotuberculosis express the well-studied adhesin molecules invasin (Inv) and YadA, capable of mediating Yop delivery (9, 54). However, Y. pestis does not express either adhesin due to an IS1541 element insertion within inv (58) and a frameshift mutation in yadA (44, 55). Y. pestis has a number of other adhesins capable of mediating host cell interaction. Both the pH 6 antigen (Psa [29, 63]) and plasminogen activator (Pla [28]) of Y. pestis have been shown to be adhesins. Psa is a tightly regulated pilus expressed at a pH of <6.7 and 37°C (52, 67) and is known to bind to β-linked galactosylated glycosphingolipids (46), low-density lipoprotein (31), and human IgG (69). Pla, expressed at 26°C but further induced at 37°C (49), is known to bind to several extracellular matrix components (23, 28, 30). The putative autotransporter, YapC, is also capable of mediating cell adhesion when it is expressed in Escherichia coli (15), as is the pilus encoded by the chaperone/usher system locus y0561-0563 (16), but neither yapC nor y0561-0563 results in significantly decreased adhesion when they are deleted from Y. pestis (15, 16).Recently, an additional adhesin of Y. pestis, Ail (adherence and invasion locus), was determined to facilitate cell binding (14, 25). Ail (encoded by y1324) is a 21.5-kDa outer membrane protein of the OmpX family that is predicted to have eight transmembrane domains and four extracellular loops extending above the surface of the bacterium (17, 65). Ail homologues include OmpX of Escherichia coli (32) and Enterobacter cloacae (61), PagC in Salmonella (53), and Opa proteins from Neisseria (10). Ail from Y. enterocolitica has been studied previously and shown to have three activities: cell adhesion, cell invasion (36), and the ability to confer serum resistance (5, 51) by binding to complement regulatory proteins (24). The residues for all three activities have been mapped to particular amino acids in the surface-exposed loops (35). Y. pseudotuberculosis Ail also confers adhesion and invasion functions (T. M. Tsang and E. S. Krukonis, unpublished data) and serum resistance (68), although the two amino acid changes between Y. pseudotuberculosis Ail and Y. pestis Ail result in decreased adhesion and invasion mediated by the former (Tsang and Krukonis, unpublished). More recently, Y. pestis Ail was also shown to mediate cell adhesion (14, 25), autoaggregation (25), and serum resistance (3, 24, 25) and to facilitate Yop delivery to host cells (14). Furthermore, Y. pestis Ail is required for virulence, as a Y. pestis Δail mutant has a >3,000-fold increase in the 50% lethal dose (14). A Y. pestis Δail mutant shows reduced binding to both epithelial and phagocytic human-derived cell lines, and in a mouse model of infection, a Y. pestis KIM5 Δail mutant colonizes host tissue to much lower levels than the parental KIM5 strain (14). Over the course of 7 days, the Δail mutant is cleared from the host (14). Together, these data demonstrate that Ail is an important adhesin that contributes to colonization and virulence.Cell adhesion is important for the establishment of a successful infection. Adhesion is also significant in Y. pestis pathogenesis because host cell contact is required for the production and translocation of the Yop effector proteins (48, 54). Bacteria can bind directly to host cell receptors (21) or use molecules like extracellular matrix (ECM) components to mediate attachment to host cells (12, 22, 30, 45, 57, 64). Common components of the cellular matrix that facilitate bacterial binding include fibronectin (22, 28, 64), collagen (23, 45), and laminin (28, 30, 45). Interactions between bacteria and ECM can lead to bridge-like attachments to host cells.Fibronectin is a large glycoprotein that is a key structural component in many tissues. This ∼220-kDa protein is commonly found as a dimer that is linked by two disulfide bonds located near the C terminus. Fibronectin is a complex molecule made up of three types of modular repeating units (43, 47). Fibronectin can bind to many substrates, including collagen (13), integrin receptors on host cells (50, 56), and heparin (60). Additionally, fibronectin contains a binding site for several bacterial pathogens at the N-terminal end of the molecule (39, 59).A number of fibronectin binding proteins on bacterial pathogens have been identified and studied, including SigB from Staphylococcus aureus (34), protein F from Streptococcus pyogenes (41), and YadA from Y. pseudotuberculosis (12, 19) and Y. enterocolitica (64). Binding of Y. pseudotuberculosis YadA to fibronectin allows Y. pseudotuberculosis to utilize β₁ integrins on the surface of host cells for invasion (12). Given the key role of Y. pestis Ail in cell adhesion, Yop delivery, and virulence, we sought to determine the component on host cells to which Ail binds.Although Ail has been studied extensively in other Yersinia species, the substrate on host cells with which Ail interacts is not known. In this study, we used a purified Y. pestis Ail to identify the extracellular matrix component, fibronectin, as a protein bound by Ail. Furthermore, Ail-mediated binding to host cells through fibronectin is important for the delivery of Yop effector proteins.     

Subcellular Localization of the Staphylococcus aureus Heme Iron Transport Components IsdA and IsdB

Staphylococcus aureus is a human pathogen that represents a tremendous threat to global public health. An important aspect of S. aureus pathogenicity is the ability to acquire iron from its host during infection. In vertebrates, iron is sequestered predominantly within heme, the majority of which is bound by hemoglobin. To acquire iron, S. aureus binds hemoglobin, removes heme, and transports it into the cytoplasm, where heme is degraded. This process is carried out by the iron-regulated surface determinant system (Isd); however, the mechanism by which hemoglobin recognition occurs is not completely understood. Here we report that the surface receptor components of the Isd system, IsdA and IsdB, physically interact with each other and are anchored to a discrete location within the cell wall. This organized localization pattern is dependent upon the iron status of the bacterium. Furthermore, we have found that hemoglobin colocalizes with IsdB at discrete sites within the cell wall. Virulence studies revealed that IsdB is required for the efficient colonization of the heart and that IsdB is differentially expressed within infected organs, suggesting that S. aureus experiences various degrees of iron starvation depending on the site of infection. These findings significantly expand our understanding of hemoglobin iron acquisition and demonstrate an orchestrated pattern of regulation and localization for the S. aureus heme iron acquisition system.Staphylococcus aureus is a commensal organism that colonizes the anterior nares of approximately 30% of the human population (26). S. aureus is also an important human pathogen that is capable of infecting virtually any site of the body (23-25). The shift from commensal colonizer to invading pathogen typically occurs upon a breakage of the skin or mucosal barrier, whereupon S. aureus employs an arsenal of virulence factors that allow it to survive within the host and cause considerable damage (29). The majority of these virulence factors are either secreted from the bacterial cell or anchored to the cell wall through the action of transpeptidases known as sortases (29, 30). The functions of cell wall-anchored proteins include adherence, immune evasion, nutrient acquisition, and resistance to antimicrobials, all processes important for the survival of S. aureus in the context of infection (6, 7, 14, 16, 19, 31-33, 35, 39, 40, 46). The contribution of cell wall-anchored proteins to the pathogenicity of S. aureus is evident through a decrease in the virulence of sortase mutants in animal models of infection (21, 22, 30, 32, 49). Among the functions carried out by sortase-anchored proteins is the acquisition of iron (31), which is a vital nutrient that is concealed from invading bacteria by host iron-sequestering proteins in a process known as nutritional immunity (2, 9).Most iron within the mammalian host is contained within the tetrapyrrole heme, the cofactor of hemoglobin (8, 11, 47). Hemoglobin is a sufficient source of iron in vitro for many bacterial pathogens including S. aureus (9, 46). S. aureus acquires iron from hemoglobin through the cooperative action of the iron-regulated surface determinant (Isd) system, which is conserved in many gram-positive pathogens (31, 44). The critical first step in this process of heme iron acquisition is hemoglobin binding to its receptors IsdB and IsdH (HarA) (12, 31, 36, 46). IsdB and IsdH then remove heme and pass it to the surface-exposed protein IsdA or to IsdC, which is embedded within the cell wall (34, 36, 50). IsdC in turn passes heme to the IsdDEF membrane transporter, which pumps heme into the cytoplasm, where it is degraded by the heme oxygenases IsdG and IsdI (34, 37, 43, 50). IsdB is required for hemoglobin binding and utilization as an iron source, while IsdA, IsdE, IsdG, and IsdI are necessary for heme iron utilization (19, 20, 28, 37, 46). The proposed model for heme iron transport through the Isd system predicts that the protein constituents of the Isd system physically interact with each other to form a molecular conduit for heme transport through the cell wall. However, the subcellular localization pattern of the Isd proteins has not been reported. In addition, it is not known if proteins of the Isd system physically engage with one another within the bacterium. Finally, the contribution of hemoglobin capture to staphylococcal virulence is incompletely defined.Here we demonstrate that IsdA and IsdB colocalize to discrete sites within the staphylococcal cell wall, and these sites correspond to regions of hemoglobin capture. IsdAB localization and subsequent hemoglobin binding are regulated by iron availability and appear to occur at the site of new cell wall formation. In support of this localization pattern, we demonstrate that IsdA and IsdB physically interact within the staphylococcal cell wall, providing direct evidence that proteins of the Isd system act as a coordinated unit to mediate hemoglobin recognition and heme iron acquisition. Finally, we report that IsdB exhibits an organ-specific regulation pattern, which corresponds to an organ-specific requirement for IsdB during the pathogenesis of S. aureus. Taken together, these results expand our understanding of the mechanism and function of hemoglobin capture by gram-positive pathogens.     

Mannheimia haemolytica and Its Leukotoxin Cause Neutrophil Extracellular Trap Formation by Bovine Neutrophils

Mannheimia haemolytica is an important member of the bovine respiratory disease complex, which is characterized by abundant neutrophil infiltration into the alveoli and fibrin deposition. Recently several authors have reported that human neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of trapping and killing pathogens. Here, we demonstrate that the leukotoxin (LKT) of M. haemolytica causes NET formation by bovine neutrophils in a CD18-dependent manner. Using an unacylated, noncytotoxic pro-LKT produced by an ΔlktC mutant of M. haemolytica, we show that binding of unacylated pro-LKT stimulates NET formation despite a lack of cytotoxicity. Inhibition of LKT binding to the CD18 chain of lymphocyte function-associated antigen 1 (LFA-1) on bovine neutrophils reduced NET formation in response to LKT or M. haemolytica cells. Further investigation revealed that NETs formed in response to M. haemolytica are capable of trapping and killing a portion of the bacterial cells. NET formation was confirmed by confocal microscopy and by scanning and transmission electron microscopy. Prior exposure of bovine neutrophils to LKT enhanced subsequent trapping and killing of M. haemolytica cells in bovine NETs. Understanding NET formation in response to M. haemolytica and its LKT provides a new perspective on how neutrophils contribute to the pathogenesis of bovine respiratory disease.Mannheimia haemolytica is a member of the bovine respiratory disease complex (BRD), causing a severe fibrinous pleuropneumonia sometimes referred to as shipping fever. The pneumonia is characterized by intense neutrophil infiltration in alveoli, intra-alveolar hemorrhage, fibrin deposition, and consolidation of the lungs (42, 56). The importance of neutrophils in the production of inflammatory mediators, recruitment of other leukocytes, and lung damage (17, 56, 67, 74) was demonstrated in calves that were depleted of neutrophils before challenge with M. haemolytica (10, 56). Neutrophil-depleted calves displayed less lung pathology than did control calves infected with M. haemolytica (10, 56). From these data, it is clear that neutrophils are a key player in the pathology of bovine pleuropneumonia; however, the mechanisms by which they contribute to host defense and tissue destruction are not clearly defined.The most important virulence factor for M. haemolytica is its leukotoxin (LKT), a 104-kDa exotoxin produced during logarithmic-phase growth (18, 32). LKT is a member of the repeats-in-toxin (RTX) toxin family of exoproteins produced by a wide variety of Gram-negative bacteria, including Escherichia coli, Actinobacillus pleuoropneumoniae, and Aggregatibacter actinomycetemcomitans (70). RTX toxins are characterized by a C-terminal glycine-rich nonapeptide repeat region (-G-G-X-G-X-D-X-U-X, where U is a hydrophobic residue) that binds calcium (Ca²⁺). The latter is required for membrane binding and cytotoxicity (30, 70). RTX toxins can insert into the plasma membrane of target cells, causing lysis and necrotic cell death (30, 70). The N-terminal domain contains amphipathic and hydrophobic domains believed to be required for pore stabilization and formation, respectively (70). More recently, it was shown that LKT also causes apoptosis via a caspase 9-dependent pathway and that LKT is internalized and transported via the cytoskeleton to mitochondria (4-6).The leukotoxin operon contains the genes lktC, lktA, lktB, and lktD (36, 37, 58). lktA encodes the inactive pro-LKT protein that is not cytotoxic until acylated (62) by the transacylase encoded by lktC. lktB and lktD encode proteins responsible for leader sequence-independent secretion of LKT from the bacterial cell (36, 37, 58). The acylated LKT then binds the CD18 chain of the β₂-integrin lymphocyte function-associated antigen 1 (LFA-1) (3, 21-26, 33, 40, 41, 44, 55, 63) on ruminant leukocytes. LKT binding to amino acids 5 to 17 of the signal sequence of CD18 is required for cell death and restricts cytotoxicity to ruminant leukocytes, because the signal sequence for CD18 is not present on mature leukocytes from other mammalian species (55). Other investigators have shown that both the pro- form and mature LKT are capable of binding CD18, although the pro-LKT does not cause cytotoxicity (62). No biological role has been assigned to the pro- form of LKT.Recently, several authors have shown that human neutrophils are able to undergo a form of cell death, called NETosis, that is distinct from apoptosis and necrosis (12, 13, 31, 51, 69). NETosis is defined as the release of nuclear DNA from an activated neutrophil into the extracellular environment, with little concomitant release of lactate dehydrogenase (LDH) (12). The extracellular DNA and associated proteins (e.g., histones) released by activated neutrophils have been termed neutrophil extracellular traps (NETs) (12). There are four steps leading to NET formation. These are neutrophil activation, nuclear envelope degradation, mixing of nuclear DNA with cytosolic proteins, and extrusion of the DNA-protein mixture from the cell (31). Treatment of human neutrophils with interleukin-8 (IL-8), phorbol 12-myristate 13-acetate (PMA), or lipopolysaccharide (LPS) causes NET formation (12, 31, 69). NET formation also occurs in response to prokaryotic and eukaryotic pathogens (12, 35, 64). To date, no bacterial exotoxin has been shown to cause NET formation.NETs are composed of extracellular DNA that is studded with antimicrobial proteins. The latter include nuclear histones and primary, secondary, and tertiary granular components such as neutrophil elastase, myeloperoxidase, lactoferrin, and gelatinase (51, 69). When neutrophils become activated and commit to NET formation, they also are capable of trapping and killing pathogens. To date, NETs have been shown to kill a variety of Gram-negative and Gram-positive bacteria, fungi, and protozoans (2, 7-9, 12, 13, 15, 19, 20, 27, 28, 31, 34, 35, 43, 50-53, 59, 64, 67, 70). Here, we examine if M. haemolytica and its LKT cause NET formation by bovine neutrophils and whether NETs are capable of trapping and killing M. haemolytica cells in vitro.     

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.     

Lipoprotein Lipase and Hydrofluoric Acid Deactivate Both Bacterial Lipoproteins and Lipoteichoic Acids,but Platelet-Activating Factor-Acetylhydrolase Degrades Only Lipoteichoic Acids

To identify the Toll-like receptor 2 ligand critically involved in infections with gram-positive bacteria, lipoprotein lipase (LPL) or hydrogen peroxide (H₂O₂) is often used to selectively inactivate lipoproteins, and hydrofluoric acid (HF) or platelet-activating factor-acetylhydrolase (PAF-AH) is used to selectively inactivate lipoteichoic acid (LTA). However, the specificities of these chemical reactions are unknown. We investigated the reaction specificities by using two synthetic lipoproteins (Pam₃CSK₄ and FSL-1) and LTAs from pneumococci and staphylococci. Changes in the structures of the two synthetic proteins and the LTAs were monitored by mass spectrometry, and biological activity changes were evaluated by measuring tumor necrosis factor alpha production by mouse macrophage cells (RAW 264.7) following stimulation. PAF-AH inactivated LTA without reducing the biological activities of Pam₃CSK₄ and FSL-1. Mass spectroscopy confirmed that PAF-AH monodeacylated pneumococcal LTA but did not alter the structure of either Pam₃CSK₄ or FSL-1. As expected, HF treatment reduced the biological activity of LTA by more than 80% and degraded LTA. HF treatment not only deacylated Pam₃CSK₄ and FSL-1 but also reduced the activities of the lipoproteins by more than 60%. Treatment with LPL decreased the biological activities by more than 80%. LPL also removed an acyl chain from the LTA and reduced its activity. Our results indicate that treatment with 1% H₂O₂ for 6 h at 37°C inactivates Pam₃CSK₄, FSL-1, and LTA by more than 80%. Although HF, LPL, and H₂O₂ treatments degrade and inactivate both lipopeptides and LTA, PAF-AH selectively inactivated LTA with no effect on the biological and structural properties of the two lipopeptides. Also, the ability of PAF-AH to reduce the inflammatory activities of cell wall extracts from gram-positive bacteria suggests LTA to be essential in inflammatory responses to gram-positive bacteria.Bacterial sepsis is a leading cause of death within intensive care units (43). Although bacterial sepsis was traditionally associated with gram-negative (Gr−) bacteria, recently, the prevalence of sepsis caused by gram-positive (Gr+) bacteria has rapidly increased (2, 3, 38). In fact, in 2000, Gr+ bacteria accounted for 52% of sepsis cases whereas Gr− bacteria accounted for only 37.6% (7, 31, 38). In bacterial sepsis, the innate immune system provides both the initial immune responses and the early inflammatory responses (1, 8, 12). Early responses to infections with Gr+ and Gr− bacteria have been shown in previous studies to involve different cytokine profiles (9, 16, 25, 51, 54). Other studies have found that infections with Gr− bacteria activate Toll-like receptor 4 (TLR4) primarily with lipopolysaccharide (LPS), a membrane component of Gr− bacteria (26, 27, 44, 53). In contrast, infections with Gr+ bacteria involve TLR2, but the nature of the key TLR2 ligand is still controversial (34, 52, 56).Two components of the cell walls of Gr+ bacteria have been proposed to be TLR2 ligands. One group of studies suggests that lipoteichoic acid (LTA) is the key ligand (10, 46, 49, 57). LTA is a polyphosphate attached to the cell membrane via a diacyl glycolipid and is an abundant component of the envelopes of Gr+ bacteria (47). Highly purified LTA, as well as its synthetic analogs, has been shown to trigger TLR2-mediated inflammatory responses (10, 15, 20, 35). However, the biological role of the LTA is unclear because it is difficult to purify natural LTA without introducing contaminants or damaging the structure of the LTA (41). Another group proposes bacterial lipoproteins as the critical ligand (22). Lipoproteins are a functionally diverse class of bacterial membrane proteins characterized by an N-terminal lipid moiety (4) and are TLR2 ligands (22-24). Although synthetic analogs of lipoproteins were found to be potent TLR2 ligands (5, 6, 42), natural lipoproteins are difficult to purify, and their properties are poorly understood.To avoid the technical difficulties involved in purification, a different investigational approach was developed. This approach uses methods to selectively inactivate either LTA or lipoproteins in bacterial culture supernatants or crude bacterial cell wall extracts (22-24, 49). LTA inactivation is usually performed with hydrofluoric acid (HF) or platelet-activating factor-acetylhydrolase (PAF-AH) (23, 48, 49), which, respectively, hydrolyzes the phosphodiester bonds in the LTA or deacylates one of its acyl chains (17, 28, 36, 55). Lipoprotein inactivation is commonly achieved by deacylation with a lipoprotein lipase (LPL) or by oxidation with hydrogen peroxide (H₂O₂) (22, 24, 62). Despite their wide use, the reaction selectivities of these methods have not been evaluated. Thus, we investigated the reaction specificities of these methods by studying the impacts of these four reactions on the biological properties as well as the chemical structures of LTA and lipoprotein analogs.     

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.     

Physical and Chemical Characterization and Immunologic Properties of Salmonella enterica Serovar Typhi Capsular Polysaccharide-Diphtheria Toxoid Conjugates

Typhoid fever remains a serious public health problem in developing countries, especially among young children. Recent studies showed more than 50% of typhoid cases are in children under 5 years old. Licensed vaccines, such as Salmonella enterica serovar Typhi capsular Vi, did not confer protection against typhoid fever for this age group. Vi conjugate, prepared by binding Vi to Pseudomonas aeruginosa recombinant exoprotein A (rEPA), induces protective levels of antibody at as young as 2 years old. Because of the lack of regulatory precedent for rEPA in licensing vaccines, we employed diphtheria toxoid (DT) as the carrier protein to accommodate accessibility in developing countries. Five lots of Vi-DT conjugates were prepared using adipic acid dihydrazide (ADH) as the linker. All 5 lots showed consistency in their physical and chemical characteristics and final yields. These Vi-DT conjugates elicited levels of IgG anti-Vi in young mice significantly higher than those in mice injected with Vi alone and induced a booster response upon reinjection. This booster effect was absent if the Vi replaced one of the two conjugate injections. Vi-DT was stable under repeated freeze-thaw (20 cycles). We plan to perform clinical evaluation of the safety and immunogenicity of Vi-DT when added to the infant combination vaccines.Typhoid fever, a serious systemic infection caused by Salmonella enterica serovar Typhi, remains a major public health problem in Central Asia, Southeast Asia, Africa, and Latin America (11, 52, 53). It was estimated that more than 21 million cases of typhoid fever and >200,000 deaths occurred in 2000 (10). The treatment of patients and management of asymptomatic carriers are becoming more difficult due to the worldwide emergence of multidrug-resistant (MDR) strains (2, 15, 29, 42, 43). Vaccination is considered the most promising strategy for the control of typhoid fever in developing countries (11, 19, 52, 53).Typhoid fever in children younger than 5 years old has often been unrecognized due to atypical clinical symptoms, difficulties in the number and volume of blood drawings, and use of less than optimal culture media (35, 46). Several studies have shown that the incidence of typhoid fever among children less than 5 years old is similar to that in school age children and young adults (14, 27, 34, 50, 51).The 3 licensed typhoid vaccines have limited efficacy, and none are suitable for young children under 5 years old. The use of heat-inactivated whole-cell vaccine was suspended in many countries because of its reactogenicity. The parenteral Vi polysaccharide and the live attenuated oral Ty21a vaccine were introduced in the late 1980s; both vaccines are well accepted and confer moderate protection (50 to 70%) in older children and adults. However, neither vaccine is licensed for routine immunization of infants (52).The Vi capsular polysaccharide is both an essential virulence factor and a protective antigen for S. Typhi (36, 38, 39). The concentration of serum IgG anti-Vi is correlated with immunity to the pathogen (22, 25, 26, 28, 36, 38, 49). However, Vi is not suitable for routine immunization of infants and young children because of its age-related immunogenicity and T-cell independence. As was shown for other capsular polysaccharides, such as Haemophilus influenzae type b (8, 37); meningococcus groups A, C, and W135; and Streptococcus pneumoniae (12, 20), Vi covalently bound with protein conferred T-cell dependence and increased immunogenicity (48-50). To date, diphtheria toxoid (DT), tetanus toxoid (TT), cholera toxins (CT), the B subunit of the heat-labile toxin (LT-B) of Escherichia coli, recombinant outer membrane protein of Klebsiella pneumoniae (rP40), and iron-regulated outer-membrane proteins (IROMPs) of S. Typhi have served as carriers for Vi polysaccharide in laboratory studies (16, 17, 32, 48-50; personal communications). An improved method was developed (24), utilizing adipic acid dihydrazide (ADH) as the linker and Pseudomonas aeruginosa recombinant exoprotein A (rEPA) as the carrier. Clinical trials of Vi-rEPA conjugates conferred 89% protection in Vietnamese children 2 to 5 years old for 46 months (23, 26, 28). The level of serum IgG anti-Vi induced by Vi-rEPA conjugates was correlated with prevention of typhoid fever in these studies (7, 21-23, 26, 28).One limitation of using rEPA as the carrier protein is the lack of regulatory precedent in licensing vaccines. In this report, five lots of Vi conjugates using DT manufactured by pharmaceutical companies in China and India were prepared (24, 48, 49). Modifications of conjugation procedures were made for the purposes of easy adoption and scale up by manufacturers. The stability of Vi-DT was studied for the feasibility of stockpiling in disaster relief.Another important aspect of conjugate vaccine implementation is the optimum immunization formulation and schedule using alternating injections of polysaccharide and conjugate. Priming or boosting effects of polysaccharide on its conjugate vaccine have been observed in infants injected with pneumococcal and meningococcal vaccines (3, 4, 31, 40). There was no consistent conclusion about various types of polysaccharides studied (6, 9, 31, 40, 41). Here, we compared the immune response of Vi polysaccharide injected before or after the administration of Vi-DT with the responses of those receiving 2 injections of Vi-DT. We also investigated the dosage effect for the purpose of better formulation.     

The Purified and Recombinant Legionella pneumophila Chaperonin Alters Mitochondrial Trafficking and Microfilament Organization

A portion of the total cellular pool of the Legionella pneumophila chaperonin, HtpB, is found on the bacterial cell surface, where it can mediate invasion of nonphagocytic cells. HtpB continues to be abundantly produced and released by internalized L. pneumophila and may thus have postinvasion functions. We used here two functional models (protein-coated beads and expression of recombinant proteins in CHO cells) to investigate the competence of HtpB in mimicking early intracellular trafficking events of L. pneumophila, including the recruitment of mitochondria, cytoskeletal alterations, the inhibition of phagosome-lysosome fusion, and association with the endoplasmic reticulum. Microscopy and flow cytometry studies indicated that HtpB-coated beads recruited mitochondria in CHO cells and U937-derived macrophages and induced transient changes in the organization of actin microfilaments in CHO cells. Ectopic expression of HtpB in the cytoplasm of transfected CHO cells also led to modifications in actin microfilaments similar to those produced by HtpB-coated beads but did not change the distribution of mitochondria. Association of phagosomes containing HtpB-coated beads with the endoplasmic reticulum was not consistently detected by either fluorescence or electron microscopy studies, and only a modest delay in the fusion of TrOv-labeled lysosomes with phagosomes containing HtpB-coated beads was observed. HtpB is the first Legionella protein and the first chaperonin shown to, by means of our functional models, induce mitochondrial recruitment and microfilament rearrangements, two postinternalization events that typify the early trafficking of virulent L. pneumophila.The gram-negative bacterium Legionella pneumophila is an intracellular parasite of amoebae (67) that has emerged as an accidental human pathogen capable of replicating in mononuclear phagocytes (38), primarily alveolar macrophages. Lung infection by L. pneumophila usually begins after the inhalation of contaminated water aerosol and manifests as an atypical pneumonia known as Legionnaires'' disease (86).The early events of L. pneumophila infection are well described at the cellular level. The first steps of infection are bacterial attachment to host cell receptors and subsequent internalization by conventional phagocytosis (27, 61, 75), coiling phagocytosis (12, 35), or macropinocytosis (84). Once internalized, L. pneumophila remains contained within a membrane-bound compartment, which is transformed into a specialized vacuole referred to as the Legionella-containing vacuole (LCV). The major cellular events of LCV conditioning include recruitment of vesicles and mitochondria (26, 33, 60), avoidance of both acidification and fusion with lysosomes (34, 37), and association with the endoplasmic reticulum (ER) (33, 42, 79, 80). Less known (or predictable) are the early changes in F-actin organization induced by L. pneumophila, which seem to be unrelated to the F-actin rearrangements required for its internalization (16, 44, 60, 78).At the molecular level, at least five bacterial gene products, RtxA, EnhC (13, 14), LpnE (58), LvhB2 (65), and HtpB (25) are involved in cell entry, confirming L. pneumophila''s flexibility in the use of alternate entry pathways. Conditioning of the LCV requires the Dot/Icm type IV secretion system of L. pneumophila, suggesting that the translocation of type IV-secreted effectors into host cells is essential for the recruitment of organelles and avoidance of acidification and fusion with lysosomes (3, 15, 53). Although a number of specific Dot/Icm effectors have been identified that mediate the sequestration of ER-derived vesicles to the LCV (19, 40, 48, 50, 63), no specific gene products have been linked to the recruitment of mitochondria or the inhibition of LCV-lysosome fusion.It has been hypothesized that the factors that mediate the internalization of L. pneumophila are preformed and may also participate in the early conditioning of the LCV (see, for example, reference 41). This hypothesis is supported by the following observations: (i) conditioning of the LCV begins within minutes after L. pneumophila internalization (18, 42, 49, 68), and (ii) antibiotic-treated legionellae (incapable of de novo protein synthesis) are not affected in their ability to attach to or enter host cells (26, 39) and resume intracellular growth immediately after removal of the antibiotic (39). It seems that L. pneumophila is prearmed to deploy a sequence of coordinated events (which follow a precise timing) immediately after making contact with a host cell, a notion also suggested by detailed microscopy studies conducted in the genetically tractable amoeba Dictyostelium discoideum (49). Moreover, some of the infection steps clearly have a short duration (49), suggesting that L. pneumophila may transiently alter a number of cellular processes. The transient nature of such effects sometimes depends on the host cell being infected. For instance, in human macrophages avoidance of both LCV acidification and fusion with lysosomes persists throughout the intracellular growth cycle (70, 85), whereas in murine macrophages, LCVs acidify and fuse with lysosomes toward the end of the L. pneumophila replicative phase (76). In contrast, other LCV conditioning processes, such as the association with mitochondria and ER, are structurally maintained (regardless of the cellular host being infected) until the replicative phase of the intracellular growth cycle has been completed (26, 33, 60).L. pneumophila HtpB is a member of the group 1 chaperonins, which includes the evolutionarily conserved and essential chaperonins of bacteria, mitochondria and plastids, with well-characterized roles in protein folding (28). The function of bacterial chaperonins, however, is not limited to protein folding. Chaperonins of bacteria can mediate adherence to mammalian cells (22), stabilize membrane lipids (81), paralyze insects (87), and activate eukaryotic signaling cascades (51, 88). The expression of HtpB is upregulated in the presence of L929 murine cells or human monocytes, and high levels of expression are maintained during the course of intracellular infection (21), leading to its accumulation in the lumen of the LCV, as shown in infected HeLa cells (24). The increased production of HtpB in L929 cells and monocytes correlates with virulence because spontaneous salt-tolerant, avirulent mutants of L. pneumophila are unable to upregulate the expression of HtpB upon contact with these host cells (21). In addition, HtpB is found in association with the L. pneumophila cytoplasmic membrane (5, 23), as well as on the bacterial cell surface (24), where it can mediate invasion of HeLa cells (25). As a L. pneumophila factor that mediates cell entry (25), and one that continues to be abundantly produced and released into the LCV after L. pneumophila internalization (24, 32), HtpB may participate in the early intracellular establishment of L. pneumophila. In the present study, we show that microbeads coated with purified HtpB (but not uncoated beads or beads coated with control proteins) are sufficient to attract mitochondria and transiently modify the organization of actin microfilaments in mammalian cells, two postinternalization events that typify the early trafficking of virulent L. pneumophila.     

The Staphylococcus aureus Epidermal Cell Differentiation Inhibitor Toxin Promotes Formation of Infection Foci in a Mouse Model of Bacteremia

Inactivation of the host GTPase RhoA by staphylococcal epidermal cell differentiation inhibitor (EDIN) exotoxins triggers the formation of large transcellular tunnels, named macroapertures, in endothelial cells. We used bioluminescent strains of Staphylococcus aureus to monitor the formation of infection foci during the first 24 h of hematogenous bacterial dissemination. Clinically derived EDIN-expressing S. aureus strains S25 and Xen36 produced many disseminated foci. EDIN had no detectable impact on infection foci in terms of histopathology or the intensity of emitted light. Moreover, EDIN did not modify the course of bacterial clearance from the bloodstream. In contrast, we show that EDIN expression promotes a 5-fold increase in the number of infection foci produced by Xen36. This virulence activity of EDIN requires RhoA ADP-ribosyltranferase activity. These results suggest that EDIN is a risk factor for S. aureus dissemination through the vasculature by virtue of its ability to promote the formation of infection foci in deep-seated tissues.Defining how pathogenic bacteria interact with the endothelium is of major relevance to evaluating the risk of severe forms of infections (21). Recent findings show that the epidermal cell differentiation inhibitor (EDIN) exotoxins trigger the formation of large transcellular tunnels, termed macroapertures, in endothelial cells of various vascular beds (5). EDINs belong to a large group of virulence factors produced by human pathogenic bacteria that target host Rho proteins (2, 4). As Rho GTPases are master regulators of the host cell actin cytoskeleton, they play a central role in controlling cell adhesion, migration, and phagocytosis (18). Moreover, Rho proteins control the formation and integrity of both intercellular adherence and tight junctions (11) and are thus major regulators of the endothelium barrier function (27, 36). Rho proteins also play essential roles in the transcellular or intercellular modes of diapedesis of leukocytes through the endothelium (6).Staphylococcal colonization of the skin and mucosa is a risk factor for bacterial translocation to underlying tissues and the bloodstream. Failure to contain the initial infection can lead to sepsis or invasion of deeper tissues, leading to endocarditis, septic arthritis, and osteomyelitis (34). Staphylococcal infections involve the combined actions of a large panel of virulence factors, promoting bacterial colonization, destruction of tissues, and immune evasion (9, 12). These bacterial virulence factors comprise secreted exotoxins and cell surface-associated factors (22). Pathogenic Staphylococcus aureus can produce enzymes such as proteases, nucleases, hyaluronidases, lipases, and cytolytic toxins. These factors participate in the destruction of host tissues and may favor bacterial dissemination in the host tissues.Several pathogenic strains of S. aureus produce EDIN type A (EDIN-A), EDIN-B, or EDIN-C (3, 15, 33, 37-39). Whereas the gene encoding EDIN-B is located on the chromosome within a pathogenicity island (15, 26, 38), genes encoding EDIN-A and EDIN-C are plasmid borne (37, 38). Strains carrying the exfoliative toxin type D gene (etd) also harbor the edin-B gene (3, 15, 38). These strains correspond to the sequence type 80 clone of Panton-Valentine leukocidin-positive community-acquired (CA) methicillin-resistant S. aureus (MRSA), which continues to spread through Europe (39) and Tunisia (3). Although genes encoding EDIN have a higher prevalence in pathogenic isolates of S. aureus (10), the contribution of EDINs to bacterial virulence remains to be defined.EDIN exotoxins translocate into the host cell cytosol from acidic compartments following macropinocytosis or after internalization into phagosomes (24). Upon reaching the cytosol, EDINs preferentially mono-ADP-ribosylate RhoA, with this modification occurring at asparagine-41 (8, 31, 35). Posttranslational modification of RhoA by ADP-ribosylation induces its tight association with RhoGDI, producing its release from membranes, where RhoA transduces signals (16, 17). Inactivation of RhoA blocks a major pathway responsible for both actin filament elongation and assembly into contractile actomyosin cables (8, 18, 28). In endothelial cells, activation of RhoA controls the formation of intercellular gaps by inducing contractile actomyosin fibers, which pull on intercellular borders (23). Thus, inactivation of RhoA by ADP-ribosylation reinforces the cohesion of adherence junctions in the endothelium (36). Nevertheless, recent advances show that inhibition of RhoA impairs endothelium barrier function by producing transcellular tunnels, referred to as macroapertures (5). Indeed, endothelial cell intoxication either by any of the three isoforms of EDIN or by infection by EDIN-producing S. aureus triggers the formation of these transcellular tunnels. Macroapertures are also formed in the endothelium lining rat arteries infected ex vivo by EDIN-producing S. aureus (5). Macroapertures unmask the fibril matrix underneath the endothelium where bacteria adhere.The capacity of EDIN to open macroapertures in vitro and ex vivo raised the possibility that this family of exotoxins might favor S. aureus hematogenous spread to deeper tissues (21). We investigated this by using bioluminescent strains of S. aureus in a mouse model of infection. Here we show that EDIN expression confers on S. aureus a greater capacity to form disseminated infection foci during bacteremia.