Protection against Intestinal Amebiasis by a Recombinant Vaccine Is Transferable by T Cells and Mediated by Gamma Interferon

We have previously shown that vaccination with purified Entamoeba histolytica Gal/GalNAc lectin or recombinant subunits can protect mice from intestinal amebiasis upon intracecal challenge. In this study, we demonstrated with adoptive-transfer experiments that this lectin vaccine protection is mediated by T cells but not serum. The cell-mediated immune (CMI) response was characterized by significant gamma interferon (IFN-γ), interleukin 12 (IL-12), IL-2, IL-10, and IL-17 production. To move toward a human vaccine, we switched to a recombinant protein and tested a range of adjuvants and routes appropriate for humans. We found that subcutaneous delivery of LecA with IDRI''s adjuvant system EM014 elicited a potent Th1-type CMI profile and provided significant protection, as measured by culture negativity (79% efficacy); intranasal immunization with cholera toxin provided 56% efficacy; and alum induced a Th2-type response that protected 62 to 68% of mice. Several antibody and CMI cytokine responses were examined for correlates of protection, and prechallenge IFN-γ⁺ or IFN-γ-, IL-2-, and tumor necrosis factor alpha-triple-positive CD4 cells in blood were statistically associated with protection. To test the role of IFN-γ in LecA-mediated protection, we neutralized IFN-γ in LecA-immunized mice and found that it abrogated the protection conferred by vaccination. These data demonstrate that CMI is sufficient for vaccine protection from intestinal amebiasis and reveal an important role for IFN-γ, even in the setting of alum.The enteric protozoan parasite Entamoeba histolytica is the causative pathogen of amebic dysentery and liver abscess that affects millions of people worldwide. Bangladeshi children experience a 40% annual incidence of E. histolytica infection (24), and evidence of prior E. histolytica infection can be detected in 8.4% of the general population in Mexico (6). Despite the availability of effective antibiotics, the World Health Organization estimates that up to 100,000 deaths occur annually, highlighting the need for alternate approaches to control amebiasis. One approach is to develop a vaccine to prevent intestinal infection (26).Several vaccine candidates for amebiasis have been proposed (48), including the serine-rich E. histolytica protein, peroxiredoxin, the EhCP112 molecule, and the galactose/N-acetyl-d-galactosamine-inhibitable lectin (Gal/GalNAc lectin). A significant body of work has focused on the latter: vaccination with either parasite-purified Gal/GalNAc lectin (10, 29, 32, 38, 40) or recombinant lectin subunits has provided protection in rodent models against amebic liver abscess and amebic colitis (29, 37, 46, 47, 53). Although these results are encouraging, two limitations remain. First, in most of these vaccine studies, the adjuvants and delivery routes are not compatible with eventual use in humans. Second, the mechanisms of amebiasis vaccine-mediated protection are still not fully understood. For instance, in the intestinal model, there was an association between the presence of an antiparasite lectin fecal immunoglobulin A (IgA) response and subsequent protection, but the association did not extend to the recombinant antigen, and fecal IgA-negative mice remained statistically protected, suggesting that other immune mechanisms exist (29). Indeed, there is increasing evidence for a role of cell-mediated immunity (CMI) in protection from intestinal amebiasis (14, 20, 28). We have found that gamma interferon (IFN-γ), the canonical Th1 cytokine, can clear intestinal amebic infection in CBA mice (21). In this study, we demonstrate for the first time that CMI plays a critical role in lectin-elicited protective immunity to intestinal amebic infection. A variety of vaccine adjuvants and delivery routes were tested for their effectiveness in protection and in eliciting CMI, and the tests demonstrated a clear role for CMI and IFN-γ in lectin-based vaccine protection.     

Characterization of Entamoeba histolytica Intermediate Subunit Lectin-Specific Human Monoclonal Antibodies Generated in Transgenic Mice Expressing Human Immunoglobulin Loci

Four fully human monoclonal antibodies (MAbs) to Entamoeba histolytica intermediate subunit lectin (Igl) were prepared in XenoMouse mice, which are transgenic mice expressing human immunoglobulin loci. Examination of the reactivities of these MAbs to recombinant Igl1 and Igl2 of E. histolytica showed that XEhI-20 {immunoglobulin G2(κ) [IgG2(κ)]} and XEhI-28 [IgG2(κ)] were specific to Igl1, XEhI-B5 [IgG2(κ)] was specific to Igl2, and XEhI-H2 [IgM(κ)] was reactive with both Igls. Gene analyses revealed that the V_H and V_L germ lines were VH3-48 and L2 for XEhI-20, VH3-21 and L2 for XEhI-28, VH3-33 and B3 for XEhI-B5, and VH4-4 and A19 for XEhI-H2, respectively. Flow cytometry analyses showed that the epitopes recognized by all of these MAbs were located on the surfaces of living trophozoites. Confocal microscopy demonstrated that most Igl1 and Igl2 proteins were colocalized on the surface and in the cytoplasm, but different localization patterns in intracellular vacuoles were also present. The preincubation of trophozoites with XEhI-20, XEhI-B5, and XEhI-H2 caused significant inhibition of the adherence of trophozoites to Chinese hamster ovary cells, whereas preincubation with XEhI-28 did not do so. XEhI-20, XEhI-B5, and XEhI-H2 were injected intraperitoneally into hamsters 24 h prior to intrahepatic challenge with E. histolytica trophozoites. One week later, the mean abscess size in groups injected with one of the three MAbs was significantly smaller than that in controls injected with polyclonal IgG or IgM isolated from healthy humans. These results demonstrate that human MAbs to Igls may be applicable for immunoprophylaxis of amebiasis.Amebiasis caused by infection with Entamoeba histolytica is one of the most problematic parasitic diseases of humans worldwide. It is estimated to result in 50 million cases of colitis and liver abscess and up to 100,000 deaths annually (54). However, an effective vaccine or chemoprophylaxis to prevent amebiasis has not been developed. The adherence of E. histolytica trophozoites to colonic mucins and various host cells is an essential event for colonization, invasion, and subsequent pathogenesis. The adherence is mediated by a galactose (Gal)- and N-acetyl-d-galactosamine (GalNAc)-inhibitable lectin (39). The lectin is a 260-kDa heterodimeric glycoprotein composed of a 170-kDa heavy subunit (Hgl) and a 31- or 35-kDa light subunit (Lgl) (38), and the Hgl is a candidate vaccine for amebiasis (22, 27, 30). We have demonstrated previously that a 150-kDa intermediate subunit (Igl), which is noncovalently associated with Hgl, also contributes to adherence (13). A mouse monoclonal antibody (MAb) specific for Igl significantly inhibits the adherence and cytotoxicity of trophozoites to mammalian cells and inhibits erythrophagocytosis (10, 48, 51). The immunization of hamsters with native Igl can inhibit amebic liver formation (11). There are two isoforms of Igl, which have 1,101 and 1,105 amino acids and are referred to as Igl1 and Igl2, respectively (9). The Igls are known to be cysteine-rich proteins containing multiple CXXC motifs, but the association between inhibitory effects and each Igl isoform is not well understood.Recent studies have shown that cellular immunity is important for the prevention of invasive amebiasis (26, 42). However, it has been reported previously that passive immunization with rabbit antiserum to a serine-rich E. histolytica protein, with human anti-E. histolytica antibodies obtained from patients with amebic liver abscesses, or with a mouse MAb to a surface lipophosphoglycan antigen inhibits amebic liver abscess formation in a severe combined immunodeficient mouse model (31, 43, 55). We have also demonstrated previously that mouse MAb to Igl can inhibit liver abscess formation in hamsters (12). Therefore, human MAbs to these antigens may be applicable to reduce mortality from amebiasis by passive immunization. Hybridoma technology has been relatively unsuccessful for the generation of human MAbs, but several new methods have recently been developed (3, 5, 53), including the use of XenoMouse mice, which are transgenic mice containing the megabase-sized human immunoglobulin loci (17, 18, 32). Several human MAbs generated using this approach are now in clinical trials (4, 14, 28, 37).In the present study, we used XenoMouse mice to generate fully human MAbs to E. histolytica Igl. Here, we report the molecular characterization of human MAbs specific for Igl1 and Igl2 of E. histolytica, and we also evaluate the effects of these human MAbs on amebic adherence in vitro and amebic liver abscess formation in hamsters.     

Development of Loop-Mediated Isothermal Amplification Assay for Detection of Entamoeba histolytica

A novel one-step, closed-tube, loop-mediated isothermal amplification (LAMP) assay for detecting Entamoeba histolytica, one of the leading causes of morbidity in developing countries, was developed. The sensitivity of the LAMP assay is 1 parasite per reaction. A total of 130 clinical samples were analyzed, and the results compared with those of conventional nested PCR to validate the practicability of this assay. No DNA was amplified from other diarrheal pathogens, such as other Entamoeba species, bacteria, and viruses. These results indicate that LAMP is a rapid, simple, and valuable diagnostic tool for epidemiological studies of amebiasis.Entamoeba histolytica is the etiologic agent of human amebiasis, which causes an estimated 40 to 50 million cases of dysentery and liver abscess and up to 100,000 deaths each year, mainly in tropical and subtropical countries (6). The detection of amebiasis in developing countries is currently dependent on microscopic examination of stool samples from patients. However, this system is at best only 10 to 60% sensitive (9, 10, 14, 22) and is incapable of distinguishing between the pathogenic species E. histolytica, the nonpathogenic species Entamoeba dispar, and the amphizoic amoeba Entamoeba moshkovskii, which infects humans sporadically (5, 7, 8). The lack of early and accurate diagnosis is a critical obstacle to clinical management of E. histolytica infection globally. Therefore, an accurate but feasible method that can be easily used in areas where amebiasis is endemic is clearly needed to confirm the identification of E. histolytica.Several new diagnostic approaches based on the detection of an E. histolytica-specific antigen and DNA had been established recently (22). The TechLab Entamoeba histolytica II kit is the only commercially available antigen test kit for specific detection of E. histolytica in feces. However, since storage or the use of preservatives destroys the antigen, it is recommended for use exclusively with fresh stool samples (21, 22). Several PCR-based assays for E. histolytica identification have subsequently been published, including single-tube multiplex reactions and nested PCR (12). The results suggest that PCR should be useful as a reference test for sensitive differentiation of E. histolytica and E. dispar (7) and could replace the TechLab enzyme-linked immunosorbent assay in areas where the pathogen occurs less frequently (23). However, the PCR assay commonly requires electrophoresis to detect the amplicons, leading to relatively high expense, including labor costs, and long turnaround time. Real-time PCR assays have also been applied to detect and quantify pathogens by continuously monitoring the amplicon formation with time (22). Nevertheless, such assays require an expensive thermal cycler with a fluorescence detector.The loop-mediated isothermal amplification (LAMP) assay was originally developed by Mori et al. (17), Nagamine et al. (18), and Notomi et al. (19) (Eiken Chemical Co., Ltd., Japan), using a set of two specifically designed inner primers and two outer primers that recognize six distinct regions of the targeted DNA. Since this reaction is performed under isothermal conditions, simple incubators, such as a water bath or heat block, are adequate for the DNA amplification. Considering these advantages, the LAMP assay could become a valuable diagnostic tool in developing countries or hospital laboratories. Therefore, the aim of this study was to develop a sensitive and specific LAMP-based method to detect E. histolytica and to evaluate this method by comparing it to a conventional nested PCR assay with clinical fecal samples.     

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.     

Genetic and antigenic diversities of major immunoreactive proteins in globally distributed Ehrlichia canis strains

The extent of knowledge regarding the diversity of globally distributed Ehrlichia canis strains has been limited to information gained from a few evolutionarily conserved genes. In this study, E. canis strains from the United States (strain Jake [US]), Brazil (strain São Paulo [BR]), and Israel (strain 611 [IS] and Ranana [IS-R]) were used to examine the antigenic and genetic diversities of four well-characterized major immunoreactive protein genes/proteins. gp36 and gp200 were the most divergent genes, and nucleotide substitutions in the gp36 tandem repeat region of the IS strain, but not the IS-R strain, resulted in two amino acid differences (S→P and P→T) in each nine-amino-acid repeat (epitope-containing region). DNA sequences of gp19 and gp140 were completely conserved in the US and BR strains, but differences were found in the Israeli strains, including two fewer tandem repeats in gp140 and a single amino acid substitution in gp19 from the IS strain. E. canis whole-cell lysates from each isolate were examined by Western immunoblotting using sera from naturally infected dogs from each country, and four major immunoreactive proteins (gp19, gp36, gp140, and gp200) were identified in each strain using protein-specific antisera. The US and BR strains exhibited highly conserved immunoreactive protein profiles, while some differences were identified in the IS strain. Sera from naturally infected Israeli dogs confirmed gene sequencing information, which demonstrated two distinct E. canis strains, defined by the gp36 gene. Conversely, gp19 was strongly reactive and present in all E. canis isolates. gp140 and gp200 were also present in all strains, although gp140 in the IS strain had two fewer tandem repeats and exhibited a smaller mass.Ehrlichia canis is a globally distributed, tick-transmitted, obligately intracellular bacterium that is the primary etiological agent of canine monocytic ehrlichiosis and has been identified as being the cause of human ehrlichiosis in patients from Venezuela (38, 39). Rickettsiosis in dogs caused by E. canis was first reported in 1935 in Algeria and was later reported in southern India and other parts of Africa in the 1940s (9, 31). Subsequently, E. canis was relatively unrecognized until it was associated with outbreaks of canine tropical pancytopenia in Singapore and Malaysia from 1963 to 1968 (51) and was identified as being the cause of an epizootic of canine tropical pancytopenia in U.S. military dogs stationed in Vietnam in late 1968 (17, 36). E. canis infections have since been well documented in the United States, Israel, Brazil, and Vietnam (1, 3, 12, 16, 20-22, 36, 49), and serologic and/or molecular evidence of infection in temperate regions where Rhipicephalus sanguineus is commonly found, including Central and South America, the Caribbean, parts of Africa, southern Europe, and southeast Asia, has also been reported (2, 5-8, 15, 18, 19, 23, 32, 33, 41, 42, 44, 50).The development of globally useful serologically and molecularly based diagnostics as well as effective vaccines for canine monocytic ehrlichiosis is dependent on an understanding of the genetic diversity of E. canis, particularly with respect to major immunoreactive proteins. Molecular characterization of evolutionarily conserved genes such as 16S rRNA has provided little information on strain diversity and suggests a high level of conservation (39, 40, 43, 47, 48). Similarly, the immunoreactive major outer membrane proteins p28 and p30 in U.S. and Venezuelan strains of E. canis appear to be highly conserved (13, 29, 30, 46), an observation that was extended to characterized E. canis strains from six human patients from Venezuela (38). Other genes such as the thio-oxidoreductase gene (dsb) and gltA were also found to be conserved in geographically dispersed strains (23, 32).The genome of E. canis has been sequenced, and a small group of acidic tandem repeat- and ankyrin repeat-containing proteins associated with host-pathogen interactions were identified (24). Several of these proteins are considered major immunoreactive proteins and have been well studied, including gp200, gp140, gp36, and gp19 (11, 25, 26, 28, 53). E. canis gp36 is an acidic serine-rich protein that contains a major antibody epitope in the tandem repeat region (11). Examination of the gp36 gene in U.S., Brazilian, and Cameroonian strains of E. canis identified variations in the numbers of tandem repeats and nucleic acid changes that resulted in four amino acid substitutions (10). However, the diversities of other major immunoreactive E. canis proteins in globally dispersed strains are not known. A homogeneous pattern of proteins reacting with E. canis dog sera from the United States, France, Israel, and the Virgin Islands by immunoblotting was previously reported (14). However, differences in protein reactivity were noted with sera collected from dogs from Italy and Zimbabwe, suggesting the potential for diversity in the antigenic composition of E. canis strains in these countries (14).The objective of this study was to determine the genetic and antigenic diversities of proteins subject to immune pressure in globally dispersed strains of E. canis. Four major immunoreactive protein genes (gp200, gp140, gp36, and gp19) were sequenced from each strain, and immunoblotting profiles for E. canis whole-cell lysates were compared. Strains from the United States and Brazil exhibited homogeneous immunoblotting patterns compared to that of the strain from Israel. Sequencing of four major immunoreactive protein genes demonstrated that U.S. and Brazilian strains were highly similar and that strains from Israel were the more divergent.     

Entamoeba histolytica Infection and Secreted Proteins Proteolytically Damage Enteric Neurons

The enteric protozoan parasite Entamoeba histolytica causes amebic colitis through disruption of the mucus layer, followed by binding to and destruction of epithelial cells. However, it is not known whether ameba infections or ameba components can directly affect the enteric nervous system. Analysis of mucosal innervations in the mouse model of cecal amebiasis showed that axon density was diminished to less than 25% of control. To determine whether amebas directly contributed to axon loss, we tested the effect of either E. histolytica secreted products (Eh-SEC) or soluble components (Eh-SOL) to an established coculture model of myenteric neurons, glia, and smooth muscle cells. Neuronal survival and axonal degeneration were measured after 48 h of exposure to graded doses of Eh-SEC or Eh-SOL (10 to 80 μg/ml). The addition of 80 μg of either component/ml decreased the neuron number by 30%, whereas the axon number was decreased by 50%. Cytotoxicity was specific to the neuronal population, since the glial and smooth muscle cell number remained similar to that of the control, and was completely abrogated by prior heat denaturation. Neuronal damage was partially prevented by the cysteine protease inhibitor E-64, showing that a heat-labile protease was involved. E. histolytica lysates derived from amebas deficient in the major secreted protease EhCP5 caused a neurotoxicity similar to that of wild-type amebas. We conclude that E. histolytica infection and ameba protease activity can cause selective damage to enteric neurons.Entamoeba histolytica is a protozoan enteric parasite of humans that colonizes the colon, where it typically causes asymptomatic luminal infections. However, in ca. 10% of individuals, the parasite invades the mucosa to cause amoebic colitis, characterized by ulcerative lesions, diarrhea, and fever and, in severe cases can disseminate to soft organs (39). Although normally seldom fatal, the consequences of ameba infection become significant in the immunosuppressed. The mechanism of infection in the intestine is complex and involves dissolution of the mucus layer by motile trophozoites, followed by adhesion and lysis of epithelial cells and invading leukocytes (8, 9).Cysteine proteases are important in the differentiation and pathogenicity of E. histolytica, and its genome contains about 50 genes coding for cysteine peptidases (36). A number of in vivo and in vitro studies have implicated cysteine protease activity as a major mechanism of cell death of infected cells, as well as degradation of the extracellular matrix and activation of the complement system (31). Although cell-cell contact is thought to be required for intestinal invasion by E. histolytica trophozoites, amebic proteins have been shown to cause cellular responses in vitro. For example, E. histolytica trophozoite-secreted products caused mucin degradation by proteolytic degradation of cysteine domains (28). In addition, incubation of secreted products and soluble proteins with cultured intestinal epithelial cells resulted in the upregulation of interleukin-8 mRNA to a similar extent as live trophozoites (11, 41). This suggests that both secreted products and direct contact serve important roles in the course of infection.The consequences to the enteric nervous system (ENS) of E. histolytica infection are unknown but may constitute an important part of its pathogenicity. The epithelial layer of the intestine is innervated by axons extending from the submucosal ganglia of the ENS. This innervation is structurally and functionally poised to respond to factors affecting the integrity of the epithelial barrier, such as amoebic invasion and the release of cysteine proteases. In addition, intestinal inflammation can lead to permanent damage to the enteric nervous system in human disease, as well as in animal models. Neuronal hypertrophy and myenteric and submucosal plexitis are among the featured characteristics observed in patients with Crohn''s disease (12), and models of colitis in the rat and other rodents show neuronal death and axonal degeneration in both the myenteric and the submucosal plexuses (24, 34). Since inflammation of the colon due to amebic invasion can resemble that seen in inflammatory bowel disease (30), we hypothesized that the enteric nervous system will also be damaged during amebic colitis.To study this, we analyzed the effect of E. histolytica infection on axon integrity in an established model of invasive murine cecal amebiasis (18). The infected intestine showed a substantial decrease in axon number compared to the control, which was inversely correlated with the extent of tissue damage. We also used an in vitro model of intestinal neurons, smooth muscle, and glia (23) and examined the effects of either amebic secreted products (Eh-SEC) or soluble components (Eh-SOL) on neuronal survival and axonal structure. We found that a population of enteric neurons was targeted by Entamoeba-derived cysteine proteases, leading to selective damage in vitro. This shows that the innervation of the mucosa by the ENS is a potential target of E. histolytica intestinal invasion.     

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.     

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.     

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

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

Plasmid-Borne Virulence-Associated Genes Have a Conserved Organization in Virulent Strains of Avian Pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) is an important respiratory pathogen of poultry. Various virulence factors are responsible for determining the pathogenicity of these strains, and it is commonly believed they are encoded on large plasmids the strains carry. This study examined a series of strains, the pathogenicity of which had previously been determined by aerosol exposure, for possession of large plasmids and found all isolates carried at least one large plasmid, regardless of the level of virulence. Virulence-associated genes carried on these plasmids were also examined, and it was shown that highly virulent strains carried at least four virulence-associated genes on their largest plasmid. Two of the virulence-associated genes were shown to be chromosomally located in a strain of intermediate virulence, while no virulence-associated genes were carried by the low-virulence strain. The organization of the virulence-associated genes was shown to be highly conserved among APEC isolates of high virulence, supporting the concept of a conserved portion of the putative virulence region that contributes to the pathogenicity of APEC strains.Avian pathogenic Escherichia coli (APEC) strains cause respiratory disease and septicemia in poultry and are economically important worldwide, causing significant mortality (13). The carriage of large plasmids is considered characteristic of APEC isolates (8), and pathogenicity is thought to be determined by virulence-associated factors encoded by them (15). These factors include serum resistance, encoded by the iss gene (14), temperature-sensitive hemagglutination, encoded by tsh (10), adhesins, the production of colicin V (ColV) and the possession of iron-scavenging mechanisms, such as aerobactin production (encoded by the iucABCD operon), and the more recently identified putative iron transport system encoded by the etsABC operon (18).Another iron acquisition system found in APEC utilizes salmochelin, a catecholate siderophore. The chromosomal iroA gene cluster that encodes this system was first found in Salmonella enterica (2) and is absent from the corresponding region of the E. coli chromosome (32), although it has been found on a transmissible plasmid from a uropathogenic E. coli isolate (34). The iroA gene cluster has been found on multiple APEC virulence plasmids (9, 17, 18, 37), and deletion studies have shown that the iroA gene cluster is required for full virulence (9).A further iron transport system, designated the sitABCD system, was first identified on a pathogenicity island in Salmonella enterica serovar Typhimurium (39), and it has been shown that sitABCD is required for full virulence of Salmonella serovar Typhimurium (16). Genomic subtraction identified the plasmid-located sitA gene from the sitABCD operon as unique to an APEC strain (32), and the sitA gene was found to be more prevalent in APEC than in commensal E. coli (18, 29, 32).The sitABCD operon occurs on APEC virulence plasmids (17, 18, 30, 37), but a sitABCD deletion mutant was still pathogenic for birds, suggesting that other iron transport systems are able to compensate for the loss of sitABCD (30).The carriage of ColV plasmids has previously been thought to be essential for virulence (3, 33, 38). However, other studies have suggested it is not the presence of the ColV gene itself but other genes that these plasmids carry that are responsible for virulence (28, 35). The well-characterized APEC virulence plasmids pAPEC-O2-ColV (18) and pAPEC-1 (9) encode ColV, while carriage of the Australian APEC virulence plasmid pVM01 does not confer production of ColV (12). Despite various ColV statuses, all three of these virulence plasmids are F-type plasmids, and hence this is potentially another way to characterize APEC virulence plasmids.SopA and SopB, which have similarity to the ParA and ParB proteins of the P1 plasmid, are thought to be essential for F-plasmid partitioning (22, 24). Detection of the genes of the sopABC locus could thus indicate the presence of a putative virulence plasmid.Strain E3 is an O-nontypeable:H28 APEC field isolate (11) that carries the 151-kb virulence plasmid pVM01 (12), which contains a virulence region with the virulence-associated genes iucA, tsh, iss, iroN, and sitA, as well as hlyF, ompT, and the etsABC operon (37). The arrangement of the virulence-associated genes around pVM01 (37) is similar to that in the plasmids pAPEC-O2-ColV from APEC strain O2 (18), pAPEC-O1-ColBM from APEC strain O1 (17), and pAPEC-1 from APEC strain χ7122 (23). Identifying a specific region that is conserved in highly virulent APEC strains will facilitate diagnosis of colibacillosis by differentiation of pathogenic strains from commensal E. coli and will also enable surveillance for pathogenic isolates in the environment of poultry.This study examined six E. coli strains, some of which were isolated from diseased birds and some of which were recovered from healthy birds (11, 36). The pathogenicity of these strains has been determined using aerosol exposure (11, 36), making this the largest known collection of APEC strains fulfilling Koch''s postulates. The series of strains includes the highly virulent strains E3, E30, and E956 and the less-virulent strains E133, E1043, and E1292. The presence of the virulence-associated genes iucA, tsh, and iss in these strains has previously been elucidated by PCR amplification (36). However, while previous studies have found many of these virulence factors to be encoded by APEC strains associated with disease (29) and have suggested that they are encoded on virulence plasmids (18), they have not conclusively determined whether they are encoded on virulence plasmids or are chromosomally encoded. Similarly, although previous studies suggest that these virulence-associated genes are consistently present in isolates from diseased birds (1, 6, 18, 21, 26, 29), no study has yet determined if these genes are consistently associated with each other.The aim of this study was to examine a series of strains of known pathogenicities for the possession of large plasmids and to determine if known virulence-associated genes from the putative virulence region were carried on them. The second objective was to investigate any association between the virulence-associated genes.     

Contribution of Autolysin and Sortase A during Enterococcus faecalis DNA-Dependent Biofilm Development

Biofilm production is a major attribute of Enterococcus faecalis clinical isolates. Although some factors, such as sortases, autolysin, and extracellular DNA (eDNA), have been associated with E. faecalis biofilm production, the mechanisms underlying the contributions of these factors to this process have not been completely elucidated yet. In this study we define important roles for the major E. faecalis autolysin (Atn), eDNA, and sortase A (SrtA) during the developmental stages of biofilm formation under static and hydrodynamic conditions. Deletion of srtA affects the attachment stage and results in a deficiency in biofilm production. Atn-deficient mutants are delayed in biofilm development due to defects in primary adherence and DNA release, which we show to be particularly important during the accumulative phase for maturation and architectural stability of biofilms. Confocal laser scanning and freeze-dry electron microscopy of biofilms grown under hydrodynamic conditions revealed that E. faecalis produces a DNase I-sensitive fibrous network, which is important for biofilm stability and is absent in atn-deficient mutant biofilms. This study establishes the stage-specific requirements for SrtA and Atn and demonstrates a role for Atn in the pathway leading to DNA release during biofilm development in E. faecalis.Biofilms are bacterial communities encased within an extracellular matrix composed of carbohydrates, proteins, and nucleic acids (10). They are an ideal environment for exchange of genetic materials, such as genes encoding virulence factors and antibiotic resistance determinants, among bacteria within a community (61, 76), while allowing the flow of water and nutrients, as well as ions and various small molecules, to bacteria within the community (8) and providing a protective shield against antibiotics, antimicrobial substances, and phagocytosis (33, 80). Development of a biofilm is a complex multistage process. It is initiated by primary adhesion of the bacteria to a substratum, which is followed by the formation of microcolonies and production of an exopolysaccharide matrix, and it finally culminates with the formation of a three-dimensional (3D) multicellular mature structure (48). Bacterial biofilms are important medically because they have been associated with the pathogenesis of chronic and device-related persistent infections, such as cystic fibrosis, urinary tract infections, and endocarditis (11).Enterococcus faecalis is a gram-positive bacterium that is a commensal of the gastrointestinal tract of healthy individuals. However, it is also an important opportunistic pathogen that is responsible for a wide variety of nosocomial infections, including endocarditis, urinary tract infections, and bacteremia (14, 17, 23, 44). E. faecalis accounts for approximately 65 to 80% of all enterococcal nosocomial infections (16). The ability of E. faecalis to adhere to and develop biofilms on medical devices, such as intravascular and urinary catheters (25, 26, 36, 37, 72), is thought to contribute to its pathogenesis. The increasing resistance of enterococci to most antibiotics, including vancomycin and linezolid (62, 63), especially in biofilms (41, 59, 78), makes treatment of enterococcal infections very difficult.Several putative virulence factors and cellular processes have been implicated in the development of biofilms in E. faecalis (36); however, very little is known about their regulation and molecular contribution to this process. One of these factors, the quorum-sensing two-component transduction signaling system encoded by the fsr locus, an important virulence factor in the pathogenesis of E. faecalis, was shown to control biofilm formation via positive regulation of the extracellular zinc metalloprotease GelE (gelatinase) and the serine protease SprE (19, 38, 54-56). These proteases were recently shown to contribute to enterococcal biofilm formation via regulation of cellular autolysis and fratricidal DNA release (70, 71). In these studies, Thomas et al. also provided the first evidence for a critical role of extracellular DNA (eDNA) in E. faecalis biofilms. eDNA is an important component of the extracellular matrix of bacterial biofilms, providing structural stability to the biofilm and protection against antimicrobials (1, 2, 42, 43, 47, 51, 52, 58, 60, 65, 77). However, the function of this macromolecule throughout the establishment and growth of E. faecalis biofilms is not well characterized.Bacterial murein hydrolases, also referred to as autolysins, have been implicated in biofilm production (58, 60). They are important contributors to cell wall growth and regulation, as well as several lytic processes (75). Two autolysins of Staphylococcus epidermidis, AtlE and Aae, are adhesins that contribute to bacterial attachment to polymeric surfaces and biofilm formation via release of eDNA (20, 21, 58). E. faecalis produces several autolysins, which were recently identified and characterized (12, 24, 35). The major E. faecalis autolysin, Atn (also known as AtlA), is an N-acetylglucosaminidase important for daughter cell separation during cellular division (12). Disruption of atn in E. faecalis resulted in increased chaining, a defect in primary attachment, and decreased biofilm production (3, 24, 31, 37, 57, 70). Recently, Thomas et al. (70) provided evidence that inactivation of this autolysin results in a decrease in DNA release similar to that of gelatinase-deficient mutants. Furthermore, they showed that GelE and SprE can differentially cleave Atn in vitro, and this processing may underlie the mechanism of cell death and DNA release in E. faecalis during biofilm formation.Due to their role in cell wall regulation, autolysins may affect the localization of cell wall-anchored proteins, which can be important for adhesion during biofilm development. In most gram-positive bacteria, membrane-anchored transpeptidase enzymes known as sortases are responsible for covalently anchoring cell surface proteins bearing an LPXTG motif to the cell wall (34). Thus far, only class A and class C sortases have been implicated in biofilm formation by and the virulence of E. faecalis (28). Deletion of srtC, also known as bps, which encodes sortase C (SrtC), resulted in a significant reduction in biofilm production and attenuation of virulence in a mouse model of urinary tract infection, unlike deletion of srtA, which had minor effects under similar conditions (28). However, given the ubiquitous nature of sortases and the limited knowledge of the activity and substrates of the sole SrtA characterized in E. faecalis, it is plausible that this enzyme may play an important role in E. faecalis physiology and/or pathogenesis under different conditions. For instance, SrtA was shown to anchor the plasmid-encoded protein Asc10, which is involved in the pheromone-induced aggregation of E. faecalis (30).In this study, we determined the contributions of E. faecalis SrtA, Atn, and eDNA to the major developmental stages leading to bacterial attachment and the establishment of mature biofilms under static and hydrodynamic conditions. E. faecalis biofilm development occurs in two key steps, which involve primary attachment followed by an accumulative stage. We demonstrate that both SrtA and Atn are required for efficient primary adherence to the substratum, while Atn and eDNA promote high levels of biofilm buildup and architectural stability during the accumulative stage. In the presence of hydrodynamic shear forces, the eDNA in E. faecalis biofilms is associated with a thick and long DNase-sensitive fibrous network that is associated with lysed cells present in the biofilm in some instances, as visualized by freeze-dry microscopy, transmission electron microscopy, and confocal laser scanning microscopy (CLSM). In contrast, atn-deficient mutants are unable to produce visible DNase-sensitive extracellular fibers under these conditions, although the biofilm remains partially sensitive to DNase treatment. These findings argue for a role for Atn in the temporal regulation of DNA release. Collectively, our data underscore the importance of SrtA, the major E. faecalis autolysin, and Atn-mediated DNA release at different stages during the establishment of E. faecalis biofilms. As a critical component of the extracellular matrix of E. faecalis biofilms, eDNA may serve as a novel target for the dissolution of these structures.     

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

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

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

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

Cohort Study of Molecular Identification and Typing of Mycobacterium abscessus,Mycobacterium massiliense,and Mycobacterium bolletii

Mycobacterium abscessus is the most common cause of rapidly growing mycobacterial chronic lung disease. Recently, two new M. abscessus-related species, M. massiliense and M. bolletii, have been described. Health care-associated outbreaks have recently been investigated by the use of molecular identification and typing tools; however, very little is known about the natural epidemiology and pathogenicity of M. massiliense or M. bolletii outside of outbreak situations. The differentiation of these two species from M. abscessus is difficult and relies on the sequencing of one or more housekeeping genes. We performed extensive molecular identification and typing of 42 clinical isolates of M. abscessus, M. massiliense, and M. bolletii from patients monitored at the NIH between 1999 and 2007. The corresponding clinical data were also examined. Partial sequencing of rpoB, hsp65, and secA led to the unambiguous identification of 26 M. abscessus isolates, 7 M. massiliense isolates, and 2 M. bolletii isolates. The identification results for seven other isolates were ambiguous and warranted further sequencing and an integrated phylogenetic analysis. Strain relatedness was assessed by repetitive-sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE), which showed the characteristic clonal groups for each species. Five isolates with ambiguous species identities as M. abscessus-M. massiliense by rpoB, hsp65, and secA sequencing clustered as a distinct group by rep-PCR and PFGE together with the M. massiliense type strain. Overall, the clinical manifestations of disease caused by each species were similar. In summary, a multilocus sequencing approach (not just rpoB partial sequencing) is required for division of M. abscessus and closely related species. Molecular typing complements sequence-based identification and provides information on prevalent clones with possible relevant clinical aspects.Rapidly growing mycobacteria (RGM) are ubiquitous organisms increasingly emerging as important human pathogens. Mycobacterium abscessus is commonly associated with wound infections and abscess formation and is the most frequent RGM causing chronic lung disease, often in immunocompromised patients (15, 22, 24). M. abscessus is also notable for its resistance to treatment and the poor clinical outcome of infection with the organism (22, 24). Within the past decade, two new species of mycobacteria closely related to M. abscessus, M. massiliense and M. bolletii, have been described (1, 3). Information on the pathogenic role of M. massiliense and M. bolletii is still scant. Recent reports have described the isolation of M. massiliense from two patients in the United States (29) and one patient in Italy (35) and, lately, the identification of M. massiliense and M. bolletii among South Korean isolates (18). Both M. massiliense and M. bolletii have also been linked to health care-associated outbreaks (8, 19, 37).The species-level identification of RGM can provide the first indication of antibiotic susceptibility and can suggest the appropriate type of patient management. For example, M. abscessus is more resistant to many antibiotics both in vivo and in vitro than M. fortuitum and M. mucogenicum, but it is usually susceptible to amikacin and clarithromycin (6, 15, 24). M. massiliense was originally reported to be distinguishable from M. abscessus and related species by its susceptibility to doxycycline (3); however, resistant isolates have since been described (19, 37), suggesting that antibiotic susceptibility results may not reliably differentiate among these closely related species.Although 16S rRNA gene sequencing has been used for the identification of nontuberculous mycobacteria (NTM), including RGM, it has limited value in distinguishing among some closely related species (9, 14). Therefore, the use of several other gene targets for the identification of mycobacteria has been proposed (2, 5, 11, 23, 25, 31, 32, 39, 41). Discrimination among M. abscessus, M. massiliense, and M. bolletii (which have identical 16S rRNA gene sequences) has proven to be difficult, with sequencing of different gene targets often providing conflicting results. Among these gene targets, partial sequencing of rpoB has increasingly been used (1, 19, 29, 37).Genotypic analysis of NTM has proven useful not only in the investigation of outbreaks and pseudo-outbreaks (38) but also in characterizing the molecular epidemiology of strains, and in assessing clonal distribution and expansion (4, 7, 13, 17). In particular, molecular typing has recently been used for the characterization of health care-related outbreaks of M. massiliense and M. bolletii (19, 37).We sought to perform a thorough molecular investigation, including strain identification and typing, for a series of 42 clinical isolates (CIs) of M. abscessus, M. massiliense, and M. bolleti from patients monitored in our institution between 1999 and 2007. A retrospective patient chart review assessed demographics, underlying conditions, and clinical history.The 42 CIs and 3 type strains were subjected to multilocus sequence analysis, including sequencing of rpoB, hsp65, secA, and the internally transcribed spacer (ITS) region. The relatedness among the isolates was assessed by use of an automated repetitive-sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE). This is the most extensive molecular characterization of non-outbreak-related isolates from patients with M. abscessus, M. massiliense, and M. bolletii infections.