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Biomedical subjects

L E Bermudez

Publications and source records attributed to L E Bermudez.

At least 19 recordsLinked to original sources
PubMed2004

Differential gene expression in mononuclear phagocytes infected with pathogenic and non-pathogenic mycobacteria.

The pathogenic mycobacteria are an insidious group of bacterial pathogens that cause the deaths of millions of people every year. One of the reasons these pathogens are so successful is that they are able to invade and replicate within host macrophages, one of the first lines of defence against intruding pathogens. In contrast, non-pathogenic mycobacteria, such as Mycobacterium smegmatis are killed rapidly by macrophages. In order to understand better the series of events that allow pathogenic mycobacteria to survive and replicate within macrophages, while the non-pathogenic mycobacteria are killed rapidly, we inoculated the human monocytic cell line U937 with pathogenic (M. tuberculosis and M. avium) and non-pathogenic (M. smegmatis) mycobacteria and monitored the expression of over 3500 genes at 4, 12 and 24 h post-inoculation using a commercially available gene array system. We observed multiple differences in the gene expression patterns of monocytes infected with pathogenic and non-pathogenic mycobacteria including genes involved in cytokine, lymphokine and chemokine production, adhesion, apoptosis, signal transduction, transcription, protein cleavage, actin polymerization and growth. We also observed differences in gene expression profiles in monocytes infected with M. tuberculosis or M. avium, indicating that there are differences in the host pathogen interactions of mononuclear phagocytes infected with different pathogenic mycobacterial species. These results increase the understanding of the mechanisms used by pathogenic mycobacteria to cause disease, the host response to these organisms, and provide new insights for antimycobacterial intervention strategies.

Gene Expression Regulation↗
PubMed2004

Topical exposure to exogenous ultraviolet-irradiated urocanic acid enhances Mycobacterium ulcerans infection in a Crl:IAF(HA)-hrBR hairless guinea-pig model of Buruli ulcer disease.

BACKGROUND: Ultraviolet radiation (UVR) pre-exposure enhances Mycobacterium ulcerans infection in the Crl:IAF(HA)-hrBR hairless guinea-pig, possibly via a photoimmunosuppressive mechanism. The trans-cis photoisomerization of epidermal urocanic acid is an important initiator of the web of events leading to photoimmunosuppression. Thus, the hypothesis tested in this paper was that topical pre-exposure to UVR-irradiated urocanic acid mixture containing cis-urocanic acid (UVR-UCA) enhances the ulcerative form of M. ulcerans infection in the Crl:IAF(HA)-hrBR hairless guinea-pig model of human Buruli ulcer disease. METHODS: Groups of six animals were subjected to daily topical treatment with either 0 (vehicle only), 0.1, 0.5 or 1 mg of trans (tUCA) or UVR-UCA (contained a cis : trans urocanic acid isomer ratio of 1 : 9) for three consecutive days. A sham treatment group was also included in the experiment. Three days following their final treatment, the guinea-pigs were intradermally infected in the right dorsal flank with 1.5 x 107 CFU of M. ulcerans in 0.1 ml of phosphate-buffered saline (PBS) and sham infected with 0.1 ml of PBS in the left dorsal flank. The resultant skin lesions were then measured over the next 21 days. At day 21 postinfection, the animals were tested for delayed-type hypersensitivity (DTH) reactivity to M. ulcerans cell fragment antigens (MCF). RESULTS: Distinct, well-demarcated, dermally situated skin nodules were present at infected, but not sham-infected, skin sites by day 3 postinfection, and the lesions progressed to frank ulcers by day 5. Between days 5 and 21, the mean lesion diameters of the UVR-UCA-treated animals were significantly (P<0.001) greater than those of the sham, vehicle only or tUCA-treated groups. UVR-UCA-treated guinea-pigs also had significantly (P<0.001) suppressed DTH responses to MCF compared with the other treatment groups. There were no significant (P>0.4) differences between the lesion sizes and DTH responses of the tUCA, vehicle only or sham treatment groups. These results demonstrate that topical exposure to UVR-UCA promotes M. ulcerans infection and suppresses DTH responses to M. uclerans antigens in infected animals. These results lend credence to the hypothesis that UVR-mediated enhancement of Buruli ulcer disease in the Crl:IAF(HA)-hrBR hairless guinea-pig model occurs via modulation of cis-urocanic acid-susceptible immune pathways.

Animals↗
PubMed2001

The white morphotype of Mycobacterium avium-intracellulare is common in infected humans and virulent in infection models.

Isolates of Mycobacterium avium-intracellulare (MAI) form multiple colony types named red-opaque, white-opaque, red-transparent (RT), and white-transparent (WT). The newly discovered WT morphotype is multidrug resistant relative to other variants in vitro. To determine whether the WT morphotype occurs in humans, 32 MAI-positive clinical samples from 2 sites were plated directly onto indicator agar without prior passage in vitro. WT was the predominant morphotype in 26 (81%) of these samples and was absent in only 2 samples. WT variants grew better than isogenic RT variants in mouse and human macrophage models of infection, and RT clones that passed through such systems underwent rapid shifts to the WT morphotype. The RT morphotype was heterogeneous with regard to infectivity. In summary, the white morphotype was common in humans and was favored in disease models. It may play an important role in the establishment and persistence of MAI infection.

Animals↗
PubMed2001

Perspective on animal models: chronic intracellular infections.

Systemic human disease caused by organisms of the Mycobacterium avium-Mycobacterium intracellulare complex (MAC) represent a chronic intracellular infection in human hosts who are usually immunocompromised. To develop improved treatment and prophylaxis, and to obtain a better understanding of pathogenesis, we studied the beige mouse (C57 beige(+)/beige(+)) challenged orally or intravenously with a human isolate that causes lethal disease in patients with AIDS (MAC 101, serovar 1). Encouraging anti-MAC studies in animals, as reviewed here, should provide the basis for considering human trials with a promising agent. The ability of an antimicrobial agent to achieve high intracellular concentrations has correlated with the in vivo activity of several specific compounds.

Animals↗
PubMed2001

Characterization and expression of secA in Mycobacterium avium.

Mycobacterium avium is both a pathogen that infects several hosts such as humans, pigs, and birds, as well as a microorganism that is encountered in environmental sources (soil and water). Protein secretion by the bacterium is likely to influence its ability to overcome adverse and competitive conditions both within or outside the host. Using a combination of cloning and information available in the databank, we characterized the secA gene from M. avium, encoding for a major preprotein translocase subunit associated with the secretion system of prokaryotics. In addition, we cloned the secA promoter sequence in a reporter construct upstream of a promoterless gfp. It was determined that the secA of M. avium shares large homology with the secA of Mycobacterium tuberculosis but not with secA of Mycobacterium leprae. secA expression was determined to be greater at logarithmic growth phase although it was also expressed at low levels during the stationary phase. secA expression was also observed when the bacteria were incubated in water as well as within human monocyte-derived macrophages and in conditions that are associated with biofilm formation. Future evaluation of the sec pathway in M. avium might provide important information about secreted proteins that are required for survival in different environments.

Adenosine Triphosphatases↗
PubMed2001

Infection of mice with Mycobacterium avium primes CD8+ lymphocytes for apoptosis upon exposure to macrophages.

Mycobacterial infection is associated with granuloma formation in which the presence of apoptosis has been recognized. The role of CD4+ T and CD8+ T cells in host protection against mycobacterial infections has been demonstrated. Previous studies, however, have shown that CD8+ T cells have a limited role in host defense against Mycobacterium avium infection, and we hypothesize that M. avium infection could lead to T cell apoptosis. To investigate this hypothesis, C57BL/6 mice were infected with M. avium strain 101, and the rate of apoptosis of splenic lymphocytes cultured ex vivo with peritoneal macrophages was determined and compared with that of controls. When exposed to infected macrophages ex vivo, splenic lymphocytes from M. avium-infected mice underwent apoptosis, as determined by the TUNEL assay. This increased T cell apoptosis above the control level was observed after 3 weeks but not after only 1 week of infection in mice. No splenic T cell apoptosis was observed when lymphocytes from Mycobacterium smegmatis-infected mice were cultured in the presence of M. smegmatis-infected peritoneal macrophages. Likewise, macrophages infected in vitro with heat-killed M. avium did not trigger T cell apoptosis. Culture of macrophages in different chamber from lymphocytes, separated by a transwell membrane, was not associated with increase of apoptosis compared with uninfected control, suggesting a requirement for direct cell-cell interactions to trigger lymphocyte apoptosis. Using a double staining TUNEL followed by anti-mouse CD4 or anti-mouse CD8 monoclonal antibodies, it was observed that only CD8+ T cells but not CD4+ T cells underwent apoptosis at 3 weeks of infection. In conclusion, M. avium infection in C57/BL6 mice for 3 weeks renders CD8+ T cells prone to apoptosis when exposed ex vivo to macrophages infected with M. avium.

Animals↗
PubMed2001

Cellular and molecular mechanisms of internalization of mycobacteria by host cells.

Mycobacteria are intracellular pathogens capable of invading mononuclear phagocytes, mucosal epithelial cells (including M cells) and Schwann cells. To enter cells, mycobacteria have been shown to interact with several molecules on macrophage and epithelial cell surfaces. This suggests adaptation to the host environment. In this review we address the strategies used by pathogenic mycobacteria to gain access to the intracellular environment.

Animals↗
PubMed2001

Activity of moxifloxacin by itself and in combination with ethambutol, rifabutin, and azithromycin in vitro and in vivo against Mycobacterium avium.

Moxifloxacin activity against Mycobacterium avium complex (MAC) was evaluated in vitro against 25 strains. The MIC was determined to range from 0.125 to 2.0 microg/ml. In addition, U937 macrophage monolayers infected with MAC strain 101 (serovar 1) were treated with moxifloxacin (0.25 to 8 microg/ml) daily, and the number of intracellular bacteria was quantitated after 4 days. Moxifloxacin showed inhibitory activity at 0.5 microg/ml and higher. To assess the activity of moxifloxacin containing regimens in vivo, we infected C57BL bg(+)/bg(+) mice with 3 x 10(7) MAC strain 101 bacteria intravenously. One week later treatment was begun with the following: (i) moxifloxacin (50 mg/kg/day or 100 mg/kg/day), ethambutol (100 mg/kg/day), or a combination of moxifloxacin and ethambutol; or (ii) moxifloxacin (100 mg/kg/day), azithromycin (200 mg/kg/day), or rifabutin (40 mg/kg/day) as oral monotherapy; or (iii) all permutations of two-drug therapy or all three drugs in combination. All groups contained at least 14 animals, and the control group received the drug vehicle. After 4 weeks, quantitative blood cultures were obtained and the number of bacteria in liver and spleen was quantitated. Moxifloxacin, ethambutol, and azithromycin were active as single agents in liver, spleen, and blood. Rifabutin showed inhibitory activity only in the blood. Two-drug combinations containing azithromycin were no more active than azithromycin alone. Similarly, the three-drug combination was not more active than azithromycin alone in the spleen. Rifabutin did not add to the activity of any other single agent or two-drug combination. Moxifloxacin at both concentrations in combination with ethambutol was significantly more active than each drug alone.

Animals↗
PubMed2001

Telithromycin is active against Mycobacterium avium in mice despite lacking significant activity in standard in vitro and macrophage assays and is associated with low frequency of resistance during treatment.

The activity of telithromycin, a new ketolide, was evaluated in vitro and in vivo against Mycobacterium avium complex (MAC) strains. The MIC of telithromycin for several M. avium isolates obtained from the blood of AIDS patients ranged from 16 to >128 microg/ml (MIC at which 90% of isolates are inhibited, >128 microg/ml), and the compound did show activity in the macrophage system at concentrations greater than 8 or 16 microg/ml, but this was dependent on the MAC strain used. Telithromycin was then administered to mice infected with MAC strain 101 for 4 weeks at doses of 100, 200, or 400 mg/kg of body weight/day. Treatment with 100 and 200 mg/kg/day was bacteriostatic, but at 400 mg/kg/day telithromycin was bactericidal for MAC strains. The frequency of the emergence of resistance to telithromycin was low despite prolonged usage (12 weeks). This study demonstrates that telithromycin is active in vivo against MAC and warrants further evaluation.

Animals↗
PubMed2001

Legionella pneumophila entry gene rtxA is involved in virulence.

Successful parasitism of host cells by intracellular pathogens involves adherence, entry, survival, intracellular replication, and cell-to-cell spread. Our laboratory has been examining the role of early events, adherence and entry, in the pathogenesis of the facultative intracellular pathogen Legionella pneumophila. Currently, the mechanisms used by L. pneumophila to gain access to the intracellular environment are not well understood. We have recently isolated three loci, designated enh1, enh2, and enh3, that are involved in the ability of L. pneumophila to enter host cells. One of the genes present in the enh1 locus, rtxA, is homologous to repeats in structural toxin genes (RTX) found in many bacterial pathogens. RTX proteins from other bacterial species are commonly cytotoxic, and some of them have been shown to bind to beta(2) integrin receptors. In the current study, we demonstrate that the L. pneumophila rtxA gene is involved in adherence, cytotoxicity, and pore formation in addition to its role in entry. Furthermore, an rtxA mutant does not replicate as well as wild-type L. pneumophila in monocytes and is less virulent in mice. Thus, we conclude that the entry gene rtxA is an important virulence determinant in L. pneumophila and is likely to be critical for the production of Legionnaires' disease in humans.

Animals↗
PubMed2001

Phenotypic and genomic analyses of the Mycobacterium avium complex reveal differences in gastrointestinal invasion and genomic composition.

Mycobacterium avium and Mycobacterium intracellulare are closely related organisms and comprise the Mycobacterium avium complex. These organisms share many common characteristics, including the ability to cause life-threatening respiratory infections in people with underlying lung pathology or immunological defects and occasionally in those with no known predisposing conditions. However, the ability to invade the mucosa of the gastrointestinal tract and cause disseminated disease in AIDS patients has not been epidemiologically linked to M. intracellulare and appears to be unique to M. avium. We compared the abilities of M. avium and M. intracellulare to tolerate the acidic conditions of the stomach, to resist the membrane-disrupting activity of cationic peptides, and to invade intestinal epithelial cells in vitro and in vivo. We observed that M. avium and M. intracellulare were both tolerant to the acidic conditions encountered in the stomach and resistant to cationic peptides. However, when strains of M. avium and M. intracellulare were examined for their ability to enter cultured human intestinal cells or mouse intestinal mucosa, we observed that M. avium could invade more efficiently than M. intracellulare. To elucidate the basis of this pathogenic difference and identify genes involved in the invasion of the intestinal mucosa, we performed chromosomal DNA subtractive hybridization using M. avium and M. intracellulare chromosomal DNAs. In all, 21 genes that were present in M. avium but absent in M. intracellulare were identified, including some that may be associated with the ability of M. avium to invade the intestinal mucosa.

Animals↗
PubMed2001

Mycobacterium avium invades the intestinal mucosa primarily by interacting with enterocytes.

Previous studies have demonstrated that Mycobacterium avium can invade intestinal epithelial cells both in vitro and in vivo. When given to mice orally, M. avium preferentially interacts with the intestinal mucosa at the terminal ileum. We evaluated the mechanism(s) of M. avium binding and invasion of the intestinal mucosa using three different systems: (i) electron microscopy following administration of M. avium into an intestinal loop in mice, (ii) quantitative comparison of the bacterial load in Peyer's patch areas of the terminal ileum versus areas that do not contain Peyer's patches, and (iii) investigation of the ability of M. avium to cause disseminated infection following oral administration using B-cell-deficient mice, lacking Peyer's patches, in comparison with C57BL/6 black mice. By all approaches, M. avium was found to invade the intestinal mucosa by interacting primarily with enterocytes and not with M cells.

Animals↗
PubMed2000

Mycobacterium avium resists exposure to the acidic conditions of the stomach.

Organisms of the Mycobacterium avium complex are common pathogens in immunosuppressed patients such as individuals with AIDS. There is evidence that in AIDS patients, the main route for M. avium infection is the gastrointestinal tract. The stomach is a formidable barrier to pathogens and the ability to resist exposure to pH lower than 3 has been shown to be a virulence determinant of enteric pathogens. Incubation of three clinical isolates of M. avium under acidic pH revealed resistance of M. avium grown both to the exponential and stationary phase at pH 2.2 for 2 h. Inhibition of protein synthesis had no effect on the acid tolerance. When the duration of the incubation at pH 2.2 was extended to 24 h, bacteria grown to the stationary phase had a significantly greater tolerance to acid than exponential phase bacteria. M. avium incubated with acid in the presence of water was significantly more resistant to pH 2.2 than M. avium in the presence of buffer. Pre-adaptation in water prior to exposure to acidic conditions was also associated with increased resistance to pH 2.2. Isoosmolarity of Hank's balanced salt solution appears to be responsible for the impaired resistance to acid between 2 and 24 h of incubation. These findings indicate that M. avium is naturally tolerant to pH<3 and that pre-adaptation under conditions similar to the conditions where M. avium is found in the environment results in increased acid resistance.

Adaptation, Physiological↗
PubMed2000

Sequence and characterization of the glyceraldehyde-3-phosphate dehydrogenase of Mycobacterium avium: correlation with an epidermal growth factor binding protein.

Mycobacterium avium is a common pathogen in AIDS patients. The extracellular environment within the granuloma shown to support mycobacterial growth is in the caseous fluid. Previous work demonstrated that the presence of human epidermal growth factor (EGF), which is found in the tissue of chronic granulomous lesions, increases the growth rate of M. avium and Mycobacterium tuberculosis. Previously, a protein capable of binding recombinant human EGF (rEGF) in a western blot was identified with homology to glyceraldehyde-3-phosphate dehydrogenase (GAP) in both M. avium and M. tuberculosis but not Mycobacterium smegmatis. Surface GAPs have been identified in group A Streptococcus, enteropathogenic Escherichia coli, Candida albicans and Schistosoma mansoni. We have cloned the gap gene of M. avium. M. avium GAP has high homology with M. tuberculosis GAP. The protein was also expressed in M. smegmatis, conveying the ability to bind rEGF, but no growth increase was observed in 7H9 broth in the presence of rEGF up to 500 ng/ml. Only one copy of the GAP gene was identified in M. avium These results contribute to the understanding of M. avium pathogenesis by characterizing its interaction with a host protein present in the site of infection.

Amino Acid Sequence↗
PubMed2000

Mycobacterium avium enters intestinal epithelial cells through the apical membrane, but not by the basolateral surface, activates small GTPase Rho and, once within epithelial cells, expresses an invasive phenotype.

Mycobacterium avium is a common pathogen in AIDS patients that is primarily (but not exclusively) acquired through the gastrointestinal tract, leading to the development of bacteraemia and disseminated disease. To cause infection through the gut, binding and invasion of the intestinal epithelial barrier are required. To characterize this process further, we determined the cell surface(s) (basolateral vs. apical membrane) that M. avium interacts with in intestinal mucosal cells in vitro. The level of binding and invasion of both HT-29 and Caco-2 intestinal cell monolayers by M. avium were similar when the assay was performed with control medium in the presence of Ca2+ (when only the apical surface was exposed), with Ca2+-depleted medium or with Ca2+-depleted medium + 1 mM EGTA (exposure of both apical and basolateral membranes), suggesting that the bacterium enters the apical surface of the epithelial lining. These observations were confirmed by assays in a transwell system and by using fluorescent microscopy. Real-time video microscopy showed that M. avium entry was not associated with membrane ruffling and the use of pharmacological inhibitors of the small GTPases demonstrated that M. avium invasion is dependent on the activation of the small GTPases Rho, but not on Rac or Cdc42. Passage of M. avium through HT-29 cells led to a phenotypic change (intracellular growth; IG) that was associated with a significantly greater (between five- and ninefold) ability to bind to and invade new monolayers of epithelial cells or macrophages when compared with the invasion by M. avium grown on agar (extracellular growth; EG). IG phenotype invasion of HT-29 cells also takes place only by the apical surface. M. avium enters intestinal epithelial cells by the apical surface and, once within the cells, changes phenotype, becoming more invasive towards both macrophages and other epithelial cells.

AIDS-Related Opportunistic Infections↗
PubMed2000

Characterization of IS666, a newly described insertion element of Mycobacterium avium.

The insertion sequence IS666 was isolated from Mycobacterium avium strain 101. IS666 is a 1474 bp insertion sequence belonging to the IS256 family, that includes IS6120 from Mycobacterium smegmatis, IS1166 and IS1295 from Rhodococcus sp. IGTS8, IST2 from Thiobacillus ferrooxidans, IS256 from Staphylococcus aureus, and ISRm3 from Rhizobium meliloti. IS666 has 24 bp imperfect inverted repeats that fit the consensus described for the family, and generates 9 bp duplications upon insertion into the host DNA with no apparent specificity in the target sequence. In contrast with its two closest homologues, IS1166 and IS6120, IS666 contains a single ORF that would codify a transposase of 434 aa. IS666 is restricted to M. avium, where it is present in 21% of the isolates in a number ranging between 1 to 7 copies.

Amino Acid Sequence↗