Cutaneous Mycobacterial Infections.

Humans encounter mycobacterial species due to their ubiquity in different environmental niches. In many individuals, pathogenic mycobacterial species may breach our first-line barrier defenses of the innate immune system and modulate the activation of phagocytes to cause disease of the respiratory tract or the skin and soft tissues, sometimes resulting in disseminated infection. Cutaneous mycobacterial infections may cause a wide range of clinical manifestations, which are divided into four main disease categories: (i) cutaneous manifestations of Mycobacterium tuberculosis infection, (ii) Buruli ulcer caused by Mycobacterium ulcerans and other related slowly growing mycobacteria, (iii) leprosy caused by Mycobacterium leprae and Mycobacterium lepromatosis, and (iv) cutaneous infections caused by rapidly growing mycobacteria. Clinically, cutaneous mycobacterial infections present with widely different clinical presentations, including cellulitis, nonhealing ulcers, subacute or chronic nodular lesions, abscesses, superficial lymphadenitis, verrucous lesions, and other types of findings. Mycobacterial infections of the skin and subcutaneous tissue are associated with important stigma, deformity, and disability. Geography-based environmental exposures influence the epidemiology of cutaneous mycobacterial infections. Cutaneous tuberculosis exhibits different clinical phenotypes acquired through different routes, including via extrinsic inoculation of the tuberculous bacilli and dissemination to the skin from other sites, or represents hypersensitivity reactions to M. tuberculosis infection. In many settings, leprosy remains an important cause of neurological impairment, deformity, limb loss, and stigma. Mycobacterium lepromatosis, a mycobacterial species related to M. leprae, is linked to diffuse lepromatous leprosy of Lucio and Latapí. Mycobacterium ulcerans produces a mycolactone toxin that leads to subcutaneous tissue destruction and immunosuppression, resulting in deep ulcerations that often produce substantial disfigurement and disability. Mycobacterium marinum, a close relative of M. ulcerans, is an important cause of cutaneous sporotrichoid nodular lymphangitic lesions. Among patients with advanced immunosuppression, Mycobacterium kansasii, the Mycobacterium avium-intracellulare complex, and Mycobacterium haemophilum may cause cutaneous or disseminated disease. Rapidly growing mycobacteria, including the Mycobacterium abscessus group, Mycobacterium chelonei, and Mycobacterium fortuitum, are increasingly recognized pathogens in cutaneous infections associated particularly with plastic surgery and cosmetic procedures. Skin biopsies of cutaneous lesions to identify acid-fast staining bacilli and cultures represent the cornerstone of diagnosis. Additionally, histopathological evaluation of skin biopsy specimens may be useful in identifying leprosy, Buruli ulcer, and cutaneous tuberculosis. Molecular assays are useful in some cases. The treatment for cutaneous mycobacterial infections depends on the specific pathogen and therefore requires a careful consideration of antimicrobial choices based on official treatment guidelines.

Human genetics of mycobacterial disease.

Mycobacterial diseases are caused by members of the genus Mycobacterium, acid-fast bacteria characterized by the presence of mycolic acids within their cell walls. Claiming almost 2 million lives every year, tuberculosis (TB) is the most common mycobacterial disease and is caused by infection with M. tuberculosis and, in rare cases, by M. bovis or M. africanum. The second and third most common mycobacterial diseases are leprosy and buruli ulcer (BU), respectively. Both diseases affect the skin and can lead to permanent sequelae and deformities. Leprosy is caused by the uncultivable M. leprae while the etiological agent of BU is the environmental bacterium M. ulcerans. After exposure to these mycobacterial species, a majority of individuals will not progress to clinical disease and, among those who do, inter-individual variability in disease manifestation and outcome can be observed. Susceptibility to mycobacterial diseases carries a human genetic component and intense efforts have been applied over the past decades to decipher the exact nature of the genetic factors controlling disease susceptibility. While for BU this search was mostly conducted on the basis of candidate genes association studies, genome-wide approaches have been widely applied for TB and leprosy. In this review, we summarize some of the findings achieved by genome-wide linkage, association and transcriptome analyses in TB disease and leprosy and the recent genetic findings for BU susceptibility.

Feline leprosy due to Candidatus 'Mycobacterium tarwinense':Further clinical and molecular characterisation of 15 previously reported cases and an additional 27 cases

OBJECTIVES: This paper, the first in a series of three on 'feline leprosy', provides a detailed description of disease referable to Candidatus 'Mycobacterium tarwinense', the most common cause of feline leprosy in Victoria, Australia. METHODS: Cases were sourced retrospectively and prospectively for this observational study, describing clinical, geographical and molecular microbiological data for cats definitively diagnosed with Candidatus 'M tarwinense' infection. RESULTS: A total of 145 cases of feline leprosy were scrutinised; 114 'new' cases were sourced from the Victorian Infectious Diseases Reference Laboratory records, veterinary pathology laboratories or veterinarians, and 31 cases were derived from six published studies. Forty-two cats were definitively diagnosed with Candidatus 'M tarwinense' infection. Typically, cats were between 3 and 11 years of age, with no gender predilection, and were generally systemically well. All had outdoor access. Most cats underwent surgical resection of lesions with adjunctive medical therapy, often utilising a combination of oral clarithromycin and rifampicin for at least 3 months. Prognosis for recovery was generally good. Resolution of lesions was not observed in the absence of treatment, but a number of untreated cats continued to enjoy an acceptable quality of life despite persistence of the disease, which extended locally but did not appear to disseminate to internal organs. Preliminary results of draft genome sequencing confirmed that the species is a member of the Mycobacterium simiae complex. CONCLUSIONS AND RELEVANCE: Candidatus 'M tarwinense', a fastidious member of the M simiae complex, is capable of causing feline leprosy with a tendency to produce lesions on the head, particularly involving the eyes and periocular skin. The disease has an indolent clinical course and generally responds favourably to therapy despite lesions often containing large numbers of organisms. Detailed genomic analysis may yield clues as to the environmental niche and culture requirement of this elusive organism. Prospective treatment trials and/or drug susceptibility testing in specialised systems would further inform treatment recommendations.

Three-dimensional in vitro models of granuloma to study bacteria-host interactions, drug-susceptibility, and resuscitation of dormant mycobacteria.

Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis, and Mycobacterium avium subsp. paratuberculosis can survive within host macrophages in a dormant state, encased within an organized aggregate of immune host cells called granuloma. Granulomas consist of uninfected macrophages, foamy macrophages, epithelioid cells, and T lymphocytes accumulated around infected macrophages. Within granulomas, activated macrophages can fuse to form multinucleated giant cells, also called giant Langhans cells. A rim of T lymphocytes surrounds the core, and a tight coat of fibroblast closes the structure. Several in vivo models have been used to study granuloma's structure and function, but recently developed in vitro models of granuloma show potential for closer observation of the early stages of host's responses to live mycobacteria. This paper reviews culture conditions that resulted in three-dimensional granulomas, formed by the adhesion of cell populations in peripheral blood mononuclear cells infected with mycobacteria. The similarities of these models to granulomas encountered in clinical specimens include cellular composition, granulomas' cytokine production, and cell surface antigens. A reliable in vitro dormancy model may serve as a useful platform to test whether drug candidates can kill dormant mycobacteria. Novel drugs that target dormancy-specific pathways may shorten the current long, difficult treatments necessary to cure mycobacterial diseases.

New pathways of protective and pathological host defense to mycobacteria.

Recent studies have uncovered new mechanisms by which the human immune system attempts to control infection and how pathogens elude these mechanisms. Mycobacterial infections are prime examples of chronic battle fields between host and pathogens. The study of tuberculosis and related mycobacterial infectious diseases such as leprosy have greatly aided in deciphering mechanisms of immune mediated protection and pathology in humans. Here we review recent insights into the role of newly discovered T cell subsets including Th17, Tregs and nonclassically restricted T cells in adaptive immunity to mycobacteria. The role of newly discovered innate immune mechanisms in tuberculosis and leprosy along with recent results from 'unbiased' genome-wide and functional genetic approaches, are deciphering critical host pathways in human infectious disease.

Mycobacterium indicus pranii downregulates MMP-9 and iNOS through COX-2 dependent and TNF-α independent pathway in mouse peritoneal macrophages in vitro.

Despite the popular belief that granulomas are innate immune mechanism to restrict mycobacterial growth, evidences suggest that granulomas facilitate growth of Mycobacterium by recruiting large numbers of uninfected macrophages to the site of infection. Matrix metalloproteinase-9 (MMP-9) has been shown to be directly involved in recruitment of macrophages at the site of infection, contributing to nascent granuloma maturation and bacterial growth. In this manuscript it is reported that heat-killed Mycobacterium indicus pranii (MIP) leads to a significant downregulation of MMP-9 in murine peritoneal macrophages in vitro. The downregulation of MMP-9 is mediated through cyclooxygenase-2 (COX-2), but independent of tumor necrosis factor-α (TNF-α). By limiting nuclear to cytoplasmic export of COX-2 and iNOS transcripts, MIP inhibits excessively-high levels of nitric oxide which can be damaging to the host during acute phases of infection. MIP has been shown to provide clinical improvement in all phases of leprosy and used for treatment of leprosy and tuberculosis.

Do mycobacteria produce endospores?

The genus Mycobacterium, which is a member of the high G+C group of Gram-positive bacteria, includes important pathogens, such as M. tuberculosis and M. leprae. A recent publication in PNAS reported that M. marinum and M. bovis bacillus Calmette-Guérin produce a type of spore known as an endospore, which had been observed only in the low G+C group of Gram-positive bacteria. Evidence was presented that the spores were similar to endospores in ultrastructure, in heat resistance and in the presence of dipicolinic acid. Here, we report that the genomes of Mycobacterium species and those of other high G+C Gram-positive bacteria lack orthologs of many, if not all, highly conserved genes diagnostic of endospore formation in the genomes of low G+C Gram-positive bacteria. We also failed to detect the presence of endospores by light microscopy or by testing for heat-resistant colony-forming units in aged cultures of M. marinum. Finally, we failed to recover heat-resistant colony-forming units from frogs chronically infected with M. marinum. We conclude that it is unlikely that Mycobacterium is capable of endospore formation.

M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells.

M. tuberculosis and M. leprae are considered to be prototypical intracellular pathogens that have evolved strategies to enable growth in the intracellular phagosomes. In contrast, we show that lysosomes rapidly fuse with the virulent M. tuberculosis- and M. leprae-containing phagosomes of human monocyte-derived dendritic cells and macrophages. After 2 days, M. tuberculosis progressively translocates from phagolysosomes into the cytosol in nonapoptotic cells. Cytosolic entry is also observed for M. leprae but not for vaccine strains such as M. bovis BCG or in heat-killed mycobacteria and is dependent upon secretion of the mycobacterial gene products CFP-10 and ESAT-6. The cytosolic bacterial localization and replication are pathogenic features of virulent mycobacteria, causing significant cell death within a week. This may also reveal a mechanism for MHC-based antigen presentation that is lacking in current vaccine strains.

The secret lives of the pathogenic mycobacteria.

Pathogenic mycobacteria, including the causative agents of tuberculosis and leprosy, are responsible for considerable morbidity and mortality worldwide. A hallmark of these pathogens is their tendency to establish chronic infections that produce similar pathologies in a variety of hosts. During infection, mycobacteria reside in macrophages and induce the formation of granulomas, organized immune complexes of differentiated macrophages, lymphocytes, and other cells. This review summarizes our understanding of Mycobacterium-host cell interactions, the bacterial-granuloma interface, and mechanisms of bacterial virulence and persistence. In addition, we highlight current controversies and unanswered questions in these areas.

Immunotherapy with Mycobacterium vaccae and peripheral blood flow in long-treated leprosy patients, a randomised, placebo-controlled trial.

OBJECTIVE: to evaluate immunotherapy as a means of improving peripheral blood flow in chronic leprosy patients. DESIGN: this was a double-blind, randomised, placebo-controlled, clinical trial. MATERIALS: heat-killed Mycobacterium vaccae 1mg plus 0.02 microg Tuberculin protein per 0.1 ml dose in borate buffer, with saline as placebo. Those studied were 92 long-treated residents of a leprosy centre in Iran, 10 of their healthy children and 10 staff members. Evaluation employed the Perimed PF2, Laser-Doppler Flowmeter, a platinum skin thermistor, and a thermal sensibility tester. METHODS: single intradermal injections of test or placebo were given to 103 patients 18 months before the blinded evaluation. Fingerpulp blood flux was measured in controlled conditions and vasomotor reflexes and skin sensation to touch, pain and heat were evaluated in 45 and 47 patients in the placebo and M. vaccae groups, respectively, and in 20 healthy control persons. RESULTS: Laser-Doppler flux, skin temperature, vasomotor reflexes and sensation were impaired in leprosy patients. Immunotherapy improved (p < 0.05) Laser-Doppler flux, skin temperature and temperature sensation. CONCLUSIONS: immunotherapy, given 18 months earlier, significantly improved blood flow and temperature sensation, in fully-treated, chronic, leprosy patients. The same principles might be employed in other conditions of reduced peripheral blood flow.

Binding of alpha2-laminins by pathogenic and non-pathogenic mycobacteria and adherence to Schwann cells.

The ability of Mycobacterium leprae to specifically bind alpha2-laminins of Schwann cells has been described recently as being an important property of the leprosy bacillus, which could explain the neural tropism of M. leprae. Therefore, the extent of the expression of alpha2-laminin-binding properties among mycobacteria was investigated. In an ELISA-based assay, all three species of Mycobacterium tested (M. tuberculosis, M. chelonae and M. smegmatis) expressed laminin-binding capacity, suggesting that the ability to bind alpha2-laminins is conserved within the genus Mycobacterium. This report also demonstrated that not only M. leprae but all the mycobacterial species tested readily interacted with the ST88-14 cells, a human schwannoma cell line, and that the addition of soluble alpha2-laminins significantly increased their adherence to these cells. These results failed to demonstrate the presence in M. leprae of a unique system based on alpha2-laminins for adherence to Schwann cells.

Interleukin-15 in mycobacterial infection of antigen-presenting cells.

Interleukin-15 (IL-15) shares many biological functions with IL-2 but also exhibits unique effects. Some of these represent the potent chemoattractant activity and expansion of distinct T-cell subsets, particularly memory T cells. IL-15 may therefore modulate the quality and quantity of cellular immune responses directed against intracellular pathogens. Immunohistochemical examination of skin lesions obtained from patients with the lepromatous or the tuberculoid form of Hansen's disease revealed intralesional IL-15 protein in both forms of the disease. In addition to Mycobacterium leprae, a number of different mycobacterial species are capable of effectively inducing IL-15 secretion in infected macrophages. In this work, increased IL-15 secretion was observed in IL-4/granulocyte-macrophage colony-stimulating factor (GM-CSF)-activated antigen-presenting cells (APC) compared with unstimulated macrophages. Immunocytological detection of intracellular IL-15 revealed that infection with different mycobacterial species resulted in different staining patterns of anti-IL-15 immunoreactive material in APC. In contrast to IL-2 or IL-7, IL-15 enhanced the cytolytic potential of immune effector cells in vitro and favoured the expansion of CD1b-restricted immune cells recognizing mycobacterial-associated antigens presented by autologous APC. IL-15 produced by infected cells in situ may represent one of the key cytokines involved in granuloma formation and may aid the augmentation of cellular immune responses directed against mycobacterial-infected cells.

Increased intracellular survival of Mycobacterium smegmatis containing the Mycobacterium leprae thioredoxin-thioredoxin reductase gene.

The thioredoxin (Trx) system of Mycobacterium leprae is expressed as a single hybrid protein containing thioredoxin reductase (TR) at its N terminus and Trx at its C terminus. This hybrid Trx system is unique to M. leprae, since in all other organisms studied to date, including other mycobacteria, both TR and Trx are expressed as two separate proteins. Because Trx has been shown to scavenge reactive oxygen species, we have investigated whether the TR-Trx gene product can inhibit oxygen-dependent killing of mycobacteria by human mononuclear phagocytes and as such could contribute to mycobacterial virulence. The gene encoding M. leprae TR-Trx was cloned into the apathogenic, fast-growing bacterium Mycobacterium smegmatis. Recombinant M. smegmatis containing the gene encoding TR-Trx was killed to a significantly lesser extent than M. smegmatis containing the identical vector with either no insert or a control M. leprae construct unrelated to TR-Trx. Upon phagocytosis, M. smegmatis was shown to be killed predominantly by oxygen-dependent macrophage-killing mechanisms. Coinfection of M. smegmatis expressing the gene encoding TR-Trx together with Staphylococcus aureus, which is known to be killed via oxygen-dependent microbicidal mechanisms, revealed that the TR-Trx gene product interferes with the intracellular killing of this bacterium. A similar coinfection with Streptococcus pyogenes, known to be killed by oxygen-independent mechanisms, showed that the TR-Trx gene product did not influence the oxygen-independent killing pathway. The data obtained in this study suggest that the Trx system of M. leprae can inhibit oxygen-dependent killing of intracellular bacteria and thus may represent one of the mechanisms by which M. leprae can deal with oxidative stress within human mononuclear phagocytes.

A mycobacterial extracytoplasmic function sigma factor involved in survival following stress.

The extracytoplasmic function (ECF) sigma factors constitute a diverse group of alternative sigma factors that have been demonstrated to regulate gene expression in response to environmental conditions in several bacterial species. Genes encoding an ECF sigma factor of Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium smegmatis, designated sigE, were cloned and analyzed. Southern blot analysis demonstrated the presence of a single copy of this gene in these species and in Mycobacterium bovis BCG, Mycobacterium leprae, and Mycobacterium fortuitum. Sequence analysis showed the sigE gene to be highly conserved among M. tuberculosis, M. avium, M. smegmatis, and M. leprae. Recombinant M. tuberculosis SigE, when combined with core RNA polymerase from M. smegmatis, reconstituted specific RNA polymerase activity on sigE in vitro, demonstrating that this gene encodes a functional sigma factor. Two in vivo transcription start sites for sigE were also identified in M. smegmatis and M. bovis BCG. Comparison of wild-type M. smegmatis with a sigE mutant strain demonstrated decreased survival of the mutant under conditions of high-temperature heat shock, acidic pH, exposure to detergent, and oxidative stress. An inducible protective response to oxidative stress present in the wild type was absent in the mutant. The mycobacterial SigE protein, although nonessential for viability in vitro, appears to play a role in the ability of these organisms to withstand a variety of stresses.

Green fluorescent protein as a marker for gene expression and cell biology of mycobacterial interactions with macrophages.

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria offers certain advantages over other bioluminescence systems because no exogenously added substrate or co-factors are necessary, and fluorescence can be elicited by irradiation with blue light without exposing the cells producing GFP to invasive treatments. A mycobacterial shuttle-plasmid vector carrying gfp cDNA was constructed and used to generate transcriptional fusions with promoters of interest and to examine their expression in Mycobacterium smegmatis and Mycobacterium bovis BCG grown in macrophages or on laboratory media. The promoters studied were: (i) ahpC from Mycoosis and Mycobacterium leprae, a gene encoding alkyl hydroperoxide reductase which, along with the divergently transcribed regulator oxyR, are homologues of corresponding stress-response systems in enteric bacteria and play a role in isoniazid sensitivity; (ii) mtrA, an M. tuberculosis response regulator belonging to the superfamily of bacterial two-component signal-transduction systems; (iii) hsp60, a previously characterized heat-shock gene from M. bovis; and (iv) tbprc3, a newly isolated promoter from M. tuberculosis. Expression of these promoters in mycobacteria was analysed using epifluorescence microscopy, laser scanning confocal microscopy, fluorescence spectroscopy, and flow cytometry. These approaches permitted assessment of fluorescence prior to and after macrophage infection, and analyses of promoter expression in individual mycobacteria and its distribution within populations of bacterial cells. Bacteria expressing GFP from a strong promoter could be separated by fluorescence-activated cell sorting from cells harbouring the vector used to construct the fusion. In addition, the stable expression of mtrA-gfp fusion in M. bovis BCG facilitated localization and isolation of phagocytic vesicles containing mycobacteria. The experiments presented here suggest that GFP will be a useful tool for analysis of mycobacterial gene expression and a convenient cell biology marker to study mycobacterial interactions with macrophages.

[Effect of heat-staining procedure on the gram staining properties of mycobacteria].

Since the establishment of Gram stain by H.C.Y. Gram in 1884, it has been widely and routinely used as an aid for differentiation of bacteria. The bacteria are divided into three categories by the staining properties; Gram-positive, -negative, and -indefinite. All the text books in the world describe that mycobacteria such as M. tuberculosis are Gram-positive. By the merest chance, however, it was found that M. lepraemurium grown in tissues was not stained by the routinely used Gram staining method. Therefore, we tried to stain some of the mycobacteria by the Gram staining procedure which is widely used at present. The results obtained indicated that the mycobacteria tested were divided into three groups; the unstainable group such as M. leprae and M. lepraemurium, the Gram-positive and difficult-to-stain group which involves such slow growing mycobacteria as M. tuberculosis, M. avium, and M. intracellulare, and the Gram-indefinite group which contains such rapid growing mycobacteria as M. phlei, M. smegmatis, and M. chelonae. However, if Gram stain is carried out by the heating procedure at the first staining step, all the mycobacteria would become Gram-positive. Therefore, we emphasize that Gram staining of mycobacteria should be performed by the heating procedure.