Genomic Degeneration and Reduction in the Fish Pathogen Mycobacterium shottsii.

Mycobacterium shottsii is a dysgonic, nonpigmented mycobacterium originally isolated from diseased striped bass (Morone saxatilis) in the Chesapeake Bay, USA. Genomic analysis reveals that M. shottsii is a Mycobacterium ulcerans/Mycobacterium marinum clade (MuMC) member, but unlike the superficially similar M. pseudoshottsii, also isolated from striped bass, it is not an M. ulcerans ecovar, instead belonging to a transitional group of strains basal to proposed "Aronson" and "M" lineages. Although phylogenetically distinct from the human pathogen M. ulcerans, the M. shottsii genome shows parallel but nonhomologous genomic degeneration, including massive accumulation of pseudogenes accompanied by proliferation of unique insertion sequences (ISMysh01, ISMysh03), large-scale deletions, and genomic reorganization relative to typical M. marinum strains. Coupled with its observed ecological characteristics and loss of chromogenicity, the genomic structure of M. shottsii is suggestive of evolution toward a state of obligate pathogenicity, as observed for other Mycobacterium spp., including M. ulcerans, M. tuberculosis, and M. leprae. IMPORTANCE Morone saxatilis (striped bass) is an ecologically and economically important finfish species on the United States east coast. Mycobacterium shottsii and Mycobacterium pseudoshottsii were originally described in the early 2000s as novel species from outbreaks of visceral and dermal mycobacteriosis in this species. Biochemical and genetic characterization place these species within the Mycobacterium ulcerans/M. marinum clade (MuMC), and M. pseudoshottsii has been proposed as an ecovar of M. ulcerans. Here, we describe the complete genome of M. shottsii, demonstrating that it is clearly not an M. ulcerans ecovar; however, it has undergone parallel genomic modification suggestive of a transition to obligate pathogenicity. As in M. ulcerans, the M. shottsii genome demonstrates widespread pseudogene formation driven by proliferation of insertion sequences, as well as genomic reorganization. This work clarifies the phylogenetic position of M. shottsii relative to other MuMC members and provides insight into processes shaping its genomic structure.

Mycobacterium xenopi multiplies within human macrophages and enhances HIV replication in vitro.

Mycobacterium xenopi can cause opportunistic infections, particularly in persons infected with human immunodeficiency virus type 1 (HIV-1). The primary focus of this effort was to determine if M. xenopi isolates could survive and grow in human peripheral blood macrophage (MPhi), and if these isolates could promote the replication of HIV-1 in vitro. M. xenopi bacilli survived and replicated 10-fold within 48 h in human MPhi while avirulent Mycobacterium smegmatis, did not grow within the MPhi. M. xenopi bacilli when cultured with peripheral blood mononuclear cells enhanced HIV-1 replication 30- and 50-fold with the macrophage-tropic HIV-1(Ba-L) and 50- and 75-fold with T-cell-tropic strain HIV-1(LAI) by 6 days post-infection when compared to M. smegmatis. The enhanced HIV replication was associated with increased production of TNF-alpha. Partial inhibition of HIV-1 induction was observed using a neutralizing anti-TNF-alpha monoclonal antibody, pentoxifylline, and matrix metalloproteinase (MMP) inhibitor I. Similar mechanisms of pathogenesis among mycobacterial species may help elucidate better treatment approaches in HIV co-infected persons.

A unique Mycobacterium species isolated from an epizootic of striped bass (Morone saxatilis).

We isolated a Mycobacterium sp. resembling Mycobacterium marinum and M. ulcerans from diseased striped bass (Morone saxatilis) during an epizootic of mycobacteriosis in the Chesapeake Bay. This isolate may represent an undescribed Mycobacterium species, based on phenotypic characteristics and comparative 16S rRNA gene sequence.

Mycobacterium ulcerans cytotoxicity in an adipose cell model.

An adipose cell (SW872) model was developed to observe cellular necrosis and apoptosis upon Mycobacterium ulcerans infection and treatment with mycobacterial exudate. Apoptosis was likely due to secreted proteins, while necrosis was likely due to mycolactone. Our data suggest that additional factors in M. ulcerans may be involved in Buruli ulcer pathogenesis.

Increased expression of Borrelia burgdorferi vlsE in response to human endothelial cell membranes.

RNA isolated from virulent Borrelia burgdorferi cells incubated with human endothelial or neurological tissue cells was subjected to subtractive hybridization using RNA from the same strain incubated in tissue culture medium alone. This RNA subtractive technique generated specific probes that hybridized to two restriction fragments (8.2 kb and 10 kb respectively) generated by EcoRI digestion of total plasmid DNA. The 10 kb EcoRI fragment localized to lp28-1 and was subsequently identified as the variable membrane protein-like sequence (vls) region, which includes an expression locus (vlsE) and 15 silent cassettes. vlsE encodes a 36 kDa outer surface protein that undergoes antigenic variation during animal infections. Primer extension analysis identified the 5' end of a transcript and a putative promoter for vlsE. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) suggested that the expression of vlsE increased when virulent B. burgdorferi cells were incubated with human tissue cells or purified cell membranes isolated from those same cell lines. A 138 bp region upstream of the vlsE region that was not reported in the genome sequence was sequenced using specific 32P end-labelled primers in a DNA cycle sequencing system at high annealing temperatures. Analysis revealed that it contained a 51 bp inverted repeat, which could form an extremely stable cruciform structure. Southern blots probed with the vlsE promoter/operator region indicated that part or all of this sequence could be found on other B. burgdorferi plasmids.

Differential expression of sigE by Mycobacterium tuberculosis during intracellular growth.

The Mycobacterium tuberculosis sigE gene encodes a sigma factor that is a member of the extracytoplasmic function subfamily of sigma factors. Using RT-PCR we demonstrated that sigE is expressed in M. tuberculosis bacilli during growth in human macrophages beginning after 30 min but before 6 h after infection through at least 5 days after infection, but that sigE is not expressed by M. tuberculosis bacteria during growth in Middlebrook 7H9 broth medium. However, sigE expression can be induced by treatment of broth cultures with hydrogen peroxide. Further, sigE is not expressed by M. tuberculosis bacilli during attachment or growth in type II pneumocytes. Using a green fluorescent protein (GFP) reporter gene fused to the sigE promoter, we observed induction of GFP expression following macrophage infection. Western blotting confirmed that sigE protein expression correlated with mRNA expression in induced systems. Analysis of the region of the M. tuberculosis genome encoding sigE suggested it is part of an operon consisting of sigE-orf1-htrA-orf2. The data presented in this report showed that sigE is differentially expressed by M. tuberculosis bacilli in macrophages and might play a role in the pathogenesis of this organism.

Changes in the virulence of Mycobacterium avium after passage through embryonated hens' eggs.

Eight-day-old embryonated hen's eggs were used as a model to study Mycobacterium avium virulence. Strains isolated from human patients caused 20-90% mortality when eggs were infected by injection of bacterial suspensions into the amniotic sac. Virulence of examined strains subsequently decreased with passage through eggs to between 0 and 40% mortality in four passages. Virulence of the egg-attenuated strains could be restored by passage through human peripheral blood mononuclear cells. The site of infection in the egg was usually the mesodermal layer of the chorioallantoic membrane. A few small granulomas containing acid-fast bacteria were seen in the liver, but not in other organs. Death of chicken embryos may have resulted from destruction of the mesodermal layer of the chorioallantoic membrane with consequent respiratory failure. PBMCs infected with less virulent egg-passaged strains of M. avium produced higher levels of tumor necrosis factor-alpha than did peripheral blood mononuclear cells infected with more virulent nonpassaged strains.

Necrosis of lung epithelial cells during infection with Mycobacterium tuberculosis is preceded by cell permeation.

Mycobacterium tuberculosis establishes infection, progresses towards disease, and is transmitted from the alveolus of the lung. However, the role of the alveolar epithelium in any of these pathogenic processes of tuberculosis is unclear. In this study, lung epithelial cells (A549) were used as a model in which to examine cytotoxicity during infection with either virulent or avirulent mycobacteria in order to further establish the role of the lung epithelium during tuberculosis. Infection of A549 cells with M. tuberculosis strains Erdman and CDC1551 demonstrated significant cell monolayer clearing, whereas infection with either Mycobacterium bovis BCG or Mycobacterium smegmatis LR222 did not. Clearing of M. tuberculosis-infected A549 cells correlated to necrosis, not apoptosis. Treatment of M. tuberculosis-infected A549 cells with streptomycin, but not cycloheximide, demonstrated a significant reduction in the necrosis of A549 cell monolayers. This mycobacterium-induced A549 necrosis did not correlate to higher levels of intracellular or extracellular growth by the mycobacteria during infection. Staining of infected cells with propidium iodide demonstrated that M. tuberculosis induced increased permeation of A549 cell membranes within 24 h postinfection. Quantitation of lactate dehydrogenase (LDH) release from infected cells further demonstrated that cell permeation was specific to M. tuberculosis infection and correlated to A549 cellular necrosis. Inactivated M. tuberculosis or its subcellular fractions did not result in A549 necrosis or LDH release. These studies demonstrate that lung epithelial cell cytotoxicity is specific to infection by virulent mycobacteria and is caused by cellular necrosis. This necrosis is not a direct correlate of mycobacterial growth or of the expression of host cell factors, but is preceded by permeation of the A549 cell membrane and requires infection with live bacilli.

Involvement of matrix metalloproteinases in human immunodeficiency virus type 1-induced replication by clinical Mycobacterium avium isolates.

The role of Mycobacterium avium isolates in modulating human immunodeficiency virus type 1 (HIV-1) replication was examined by use of an in vitro, resting T cell system. Two human clinical isolates (serotypes 1 and 4) but not an environmental M. avium isolate (serotype 2) enhanced HIV-1 replication. The M. avium-induced HIV-1 replication was not associated with cell activation or differential cytokine production or utilization. Addition of matrix metalloproteinase (MMP) inhibitors and their in vivo regulators, tissue inhibitors of metalloproteinases-1 and -2, abrogated M. avium-induced HIV-1 replication 80%-95%. The MMP inhibitors did not have any effect on the HIV-1 protease activity, suggesting that they may affect cellular processes. Furthermore, MMP-9 protein was differentially expressed after infection with clinical M. avium isolates and paralleled HIV-1 p24 production. Collectively, these data suggest that M. avium-induced HIV-1 replication is mediated, in part, through the induction of MMP-9.

Observed differences in virulence-associated phenotypes between a human clinical isolate and a veterinary isolate of Mycobacterium avium.

Mycobacterium avium, the most common opportunistic pathogen in patients with AIDS, is frequently isolated from a variety of environmental sources, but rarely can these environmental isolates be epidemiologically linked with isolates known to cause human disease. Using a number of in vitro tissue culture assays, we found significant pathogenic differences between a serotype 4 human clinical M. avium isolate and a serotype 2 veterinary isolate. Cell association of the patient strain with a human intestinal cell line was 1.7 times that of the veterinary strain. Growth of this clinical strain in human peripheral blood mononuclear cell-derived macrophages increased from 12-fold higher than that of the veterinary isolate after 2 days to 200-fold higher after 4 days. By the conclusion of each experiment, lysis of all examined host cell types and accumulation of cell debris were observed in infections with the human isolate, but monolayers remained relatively intact in the presence of the animal isolate. The two strains also differed in the ability to stimulate human immunodeficiency virus replication in coinfected host cells, with p24 antigen levels after 6 days threefold higher in the cells coinfected with the clinical strain than in those infected with the veterinary strain. If the genetic differences responsible for the phenotypes observed in these assays can be identified and characterized, it may be possible to determine which M. avium strains in the environment are potential human pathogens.

Emergence of a unique group of necrotizing mycobacterial diseases.

Although most diseases due to pathogenic mycobacteria are caused by Mycobacterium tuberculosis, several other mycobacterial diseases-caused by M. ulcerans (Buruli ulcer), M. marinum, and M. haemophilum-have begun to emerge. We review the emergence of diseases caused by these three pathogens in the United States and around the world in the last decade. We examine the pathophysiologic similarities of the diseases (all three cause necrotizing skin lesions) and common reservoirs of infection (stagnant or slow-flowing water). Examination of the histologic and pathogenic characteristics of these mycobacteria suggests differences in the modes of transmission and pathogenesis, though no singular mechanism for either characteristic has been definitively described for any of these mycobacteria.

An in vitro tissue culture bilayer model to examine early events in Mycobacterium tuberculosis infection.

A tissue culture bilayer system that mimics some aspects of early alveolar infection by Mycobacterium tuberculosis was developed. This model incorporates human lung epithelial type II pneumocyte (A549) (upper chamber) and endothelial cell (lower chamber) layers separated by a microporous membrane. This construction makes it possible to observe and quantify the passage of bacteria through the two layers, to observe the interaction of the bacteria with the various cell types, and to examine the basic mechanisms of immune cell recruitment to the site of infection. After 10(7) organisms were added to the upper chamber we microscopically observed large numbers of bacteria attached to and within the pneumocytes and we determined by viable-cell counting that a small percentage of the inoculum (0.02 to 0.43%) passed through the bilayer into the lower chamber. When peripheral blood mononuclear cells were added to the lower chamber, microscopic examination indicated a migration of the mononuclear cells through the bilayer to the apical surface, where they were seen associated with the mycobacteria on the pneumocytes. The added complexity of the bilayer system offers an opportunity to define more precisely the roles of the various lung cell types in the pathogenesis of early tuberculosis.

HeLa cells as a model to study the invasiveness and biology of Legionella pneumophila.

HeLa cells were established as a model system to study the invasiveness and biology of Legionella pneumophila. In this model, invasion could be distinguished from adherence; virulent strains of L. pneumophila were adherent and invasive, whereas nonvirulent strains were adherent but poorly invasive. Invasion was rapid and did not require de novo bacterial protein synthesis, suggesting that the invasion factor is constitutively expressed by virulent strains. Entry into HeLa cells required actin polymerization and an intact microtubule cytoskeleton and was only moderately inhibited by the presence of 100 mM glucose or galactose. Intracellular replication of virulent L. pneumophila took place in ribosome-studded complex endosomes and led to the formation of free bacteria-laden vesicles presumably released from lysed HeLa cells. These free vesicles (referred to as mature vesicles) were isolated in continuous density gradients of Percoll. The bacteria contained in the isolated mature vesicles had a unique envelope structure and were highly adherent to HeLa cells, characteristics that correlated with a bright red appearance after the Giménez stain (Giménez positive). Plate-grown legionellae and replicating legionellae, harboured in complex endosomes, displayed a typical Gram-negative envelope and stained green after the Giménez stain (Giménez negative). Chronically infected cultures of HeLa cells were also established that may be a useful tool for studying long-term interactions between virulent L. pneumophila and mammalian cells. HeLa cells constitute a valuable model system that offers unique opportunities to study parasite-directed endocytosis, as well as stage specific-parasite interactions.

In search of virulence factors of human bacterial disease.

Traditional genetic techniques and a variety of animal and tissue-culture model systems have sustained the study of bacterial virulence mechanisms for several decades. However, the recent application of newly developed molecular and cellular techniques has brought our understanding of bacterial pathogenesis to new heights by permitting the identification and analysis of previously unknown constitutively and differentially expressed virulence-associated factors.