Rv2617c and P36 are virulence factors of pathogenic mycobacteria involved in resistance to oxidative stress.

In this study, we characterized the role of Rv2617c in the virulence of Mycobacterium tuberculosis. Rv2617c is a protein of unknown function unique to M. tuberculosis complex (MTC) and Mycobacterium leprae. In vitro, this protein interacts with the virulence factor P36 (also named Erp) and KdpF, a protein linked to nitrosative stress. Here, we showed that knockout of the Rv2617c gene in M. tuberculosis CDC1551 reduced the replication of the pathogen in a mouse model of infection and favored the trafficking of mycobacteria to phagolysosomes. We also demonstrated that Rv2617c and P36 are required for resistance to in vitro hydrogen peroxide treatment in M. tuberculosis and Mycobacterium bovis, respectively. These findings indicate Rv2617c and P36 act in concert to prevent bacterial damage upon oxidative stress.

Incidence of mycobacterial disorders in Indian adult male service population followed for over two decades.

BACKGROUND: The comprehensive epidemiology of mycobacterial disorders is scarce from our country. The incidence of Tuberculosis (TB) and Leprosy in a cohort of military personnel followed for a long duration was evaluated in this study. METHODS: The data for this descriptive epidemiologic study was derived from the electronic medical records (EMR) data of the service personnel enrolled between 1990 and 2015. They were recruited between the ages of 17 and 18 years in good health and their morbidity data was derived from the medical records. The incidence rate (IR) was calculated as per person-years (py) using appropriate statistical methods. RESULTS: The study population includes 51,217 participants (median age 33 years, range 17-54) with a mean follow up of 12.5 years. Yearly evaluation of the data gave a cumulative follow up duration of 613,925py. A total of 530 patients developed TB, giving an IR of 86.3 per 100,000 person years (95% CI 79.2-93.9). Leprosy was diagnosed in 59 cases giving an IR of 9.6 per 100,000py (95% CI 7.4-12.3). Pulmonary (71%) and pleural (24%) locations were the most common sites of the TB infection. The data about the contribution of the mycobacterial disorders towards the mortality and the subtypes of leprosy was not available in the EMR. CONCLUSION: Low IR of mycobacterial disorders was observed in this study when compared with the previous reports. Healthy lifestyle and good socioeconomic status could explain the low IR of mycobacterial disorders in the military personnel.

Case Report: Histoplasmosis in Himachal Pradesh (India): An Emerging Endemic Focus.

We describe four cases of histoplasmosis indigenous to Himachal Pradesh (India) that will be of considerable public health interest. A 48-year-old human immunodeficiency virus (HIV)-negative man with cervical and mediastinal lymphadenopathy, hepatosplenomegaly, adrenal mass, and bone marrow involvement was treated as disseminated tuberculosis without benefit. Progressive disseminated histoplasmosis was diagnosed from the fungus in smears from adrenal mass. Another 37-year-old HIV-positive man was on treatment of suspected pulmonary tuberculosis. He developed numerous erythema nodosum leprosum-like mucocutanous lesions accompanied by fever, generalized lymphadenopathy, and weight loss. Pulmonary histoplasmosis with cutaneous dissemination was diagnosed when skin lesions showed the fungus in smears, histopathology, and mycologic culture. Both were successfully treated with amphotericin B/itraconazole. Third patient, a 46-year-old HIV-negative man, had oropharyngeal lesions, cervical lymphadenopathy, intermittent fever, hepatosplenomegaly, and deteriorating general health. Progressive disseminated oropharyngeal histoplasmosis was diagnosed from the fungus in smears and mycologic cultures from oropharyngeal lesions and cervical lymph node aspirates. He died despite initiating treatment with oral itraconazole. Another 32-year-old man 3 months after roadside trauma developed a large ulcer with exuberant granulation tissue over left thigh without evidence of immunosuppression/systemic involvement. He was treated successfully with surgical excision of ulcer under amphotericin B/itraconazole coverage as primary cutaneous histoplasmosis confirmed pathologically and mycologically. A clinical suspicion remains paramount for early diagnosis of histoplasmosis particularly in a nonendemic area. Most importantly, with such diverse clinical presentation and therapeutic outcome selection of an appropriate and customized treatment schedule is a discretion the treating clinicians need to make.

Clofazimine has delayed antimicrobial activity against Mycobacterium tuberculosis both in vitro and in vivo.

OBJECTIVES: The anti-leprosy drug clofazimine has been shown to have antimicrobial activity against Mycobacterium tuberculosis and has been associated with treatment-shortening activity in both clinical and preclinical studies of TB chemotherapy. However, a reported lack of early bactericidal activity (EBA) in TB patients has raised questions regarding the usefulness of clofazimine as an anti-TB drug. Our objective was to systematically evaluate the EBA of clofazimine in vitro and in vivo to provide insight into how and when this drug exerts its antimicrobial activity against M. tuberculosis. METHODS: We evaluated the 14 day EBA of clofazimine (i) in vitro at concentrations ranging from 4 times below to 4 times above the MIC for M. tuberculosis and (ii) in vivo in infected BALB/c mice at doses ranging from 1.5 to 100 mg/kg/day, and serum clofazimine levels were measured. In both experiments, isoniazid was used as the positive control. RESULTS: In vitro, clofazimine, at any concentration tested, did not exhibit bactericidal activity during the first week of exposure; however, in the second week, it exhibited concentration-dependent antimicrobial activity. In vivo, clofazimine, at any dose administered, did not exhibit bactericidal activity during the first week, and limited antimicrobial activity was observed during the second week of administration. While serum clofazimine levels were clearly dose dependent, the antimicrobial activity was not significantly related to the dose administered. CONCLUSIONS: Our data suggest that clofazimine's delayed antimicrobial activity may be due more to its mechanism of action rather than to host-related factors.

Detection of Mycobacterium leprae by PCR testing of sputa from a patient with pulmonary cryptococcus coinfection in northern Australia.

A case of fever, sepsis, and chest lesions evident on a computed tomography scan of an indigenous man in northern Australia following burns to the feet is described. Sputum PCR testing revealed Mycobacterium leprae, and a fine-needle aspirate of the chest lesions demonstrated Cryptococcus coinfection.

Nanobiotechnological approaches to delivery of DNA vaccine against fungal infection.

Vaccines play an essential role in keeping humans healthy. Innovative approaches to their use include the utilization of plasmid DNA encoding sequences to express foreign antigens. DNAhsp65 from Mycobacterium leprae is suitable for this purpose due to its ability to elicit a powerful immune response. Controlled release systems represent a promising approach to delivering vaccines. In this work, we used liposomes or PLGA systems to deliver DNAhsp65 to treat the pulmonary fungal infection Paracoccidioidomycosis. Both formulations modulated a protective immune response and reduced the pulmonary fungal burden even in the groups receiving less than four times the amount of the DNAhps65 entrapped within the nanoparticles. Although both systems had the same effective therapeutic results, the advantage of the liposome formulation was that it was administered intranasally, which may be more easily accepted by patients. These systems are a great alternative to be considered as adjuvant vaccine therapy for systemic mycosis.

[Molecular mechanisms of dormancy and drug tolerance in mycobacteria].

Instead of rapid multiplication, pathogenic mycobacteria, such as Mycobacterium tuberculosis are likely to have acquired slow but long life. Host immunity affords desirable non-competitive environment for M tuberculosis in human lungs, where this pathogen slowly grows or arrests growing, which avoids rapid loss of living places. Mycobacterial DNA-binding protein 1 (MDP1), a unique histone-like protein associating mycobacterial GC-rich DNA, has pivotal role in realizing such slow life and pathogenesis including drug tolerance to isoniazid.

Expression of CXCL10 (IP-10) and CXCL11 (I-TAC) chemokines during Mycobacterium tuberculosis infection and immunoprophylaxis with Mycobacterium indicus pranii (Mw) in guinea pig.

Mycobacterium indicus pranii (earlier known as Mycobacterium w) has been used as an immunmodulatory agent in leprosy and tuberculosis by mediating the release of various cytokines and chemokines. CXCL10 (IP-10) and CXCL11 (I-TAC) chemokines are involved in T-cell migration and stimulation of natural killer cells in Mycobacterium tuberculosis infection. In this study, the effect of heat killed M. indicus pranii (alone and in conjunction with chemotherapy) on disease progression was determined by colony forming units (CFUs) in guinea pig lung following their aerosol infection and the expression levels of CXCL10 and CXCL11 were studied by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) and in situ RT-PCR. Four groups of animals included; infection only (Rv), immunoprophylaxis (RvMw), chemotherapy (RvCh) and combination of immunoprophylaxis with chemotherapy (RvChMw). In the group where immunoprophylaxis was given in combination with chemotherapy, the CFU counts reduced significantly at 4th week post-infection as compared to animals that received immunoprophylaxis or chemotherapy alone. At the same time, all groups of animals had elevated expression of CXCL 10 which was significantly high only in animals that received Mw with or without chemotherapy. Unlike to CXCL 10, study demonstrated suppressed expression CXCL 11 in both immunoprophylaxis as well as chemotherapy groups that became up-regulated in synergistic response of immunoprophylaxis and chemotherapy. Taken together, data indicates that the expression of CXCL10 and CXCL11 positively correlates with anti-tubercular treatment (at least with combination of immunoprophylaxis and chemotherapy). Therefore, prior immunization with Mw appears to be a good immunomodulator for release of chemokines and augments the effect of chemotherapy.

Incorporation of immunostimulatory motifs in the transcribed region of a plasmid DNA vaccine enhances Th1 immune responses and therapeutic effect against Mycobacterium tuberculosis in mice.

T-helper type 1 (Th1) immune response is involved in the development of protective immunity against Mycobacterium tuberculosis. Thus, an increase in Th1 and cellular immune responses should lead to enhanced anti-mycobacterial activity. In this study, we aimed to improve Th1 immune responses to a DNA vaccine by adding potentially immunostimulatory nucleotide sequences into the transcribed region downstream of the antigen. The Mycobacterium leprae gene for hsp65, codon-optimized for expression in mammalian cells, was inserted into pVAX1 with and without 3'-sequences containing CpG and dsRNA motifs. When the plasmid contained both motifs, transfected murine macrophage-like RAW264.7 cells showed markedly increased levels of mRNA for immune molecules of Th1 (IFN-α, IL-12) and Th17 (IL-17, IL-23 and IL-6) responses and for T cell co-stimulatory molecules (CD80 and CD86) but not for a Th2 response (IL-4 and IL-10). Immunized mice showed substantially increased serum anti-Hsp65 IgG2a antibody levels and IFN-γ production by spleen cells, confirming enhancement of the Th1 response in vivo. Furthermore, when non-vaccinated mice were infected with H37Rv by low-dose aerosol challenge, and then 4 weeks later were treated with plasmids by intramuscular injection, the mice that had been treated with plasmids containing immunostimulatory motifs showed an enhanced reduction in mycobacterial loads in lung and spleen. We conclude that DNA vaccines may be made more highly immunogenic and more effective for treatment by including transcribed stimulatory sequences.

A subunit vaccine based on biodegradable microspheres carrying rHsp65 protein and KLK protects BALB/c mice against tuberculosis infection.

Of the hundreds of new tuberculosis (TB) vaccine candidates, some have therapeutic value in addition to their prophylactic properties. This is the case for the DNA vaccine encoding heat-shock protein 65 (DNAhsp65) from Mycobacterium leprae. However, there are concerns about the use of DNA vaccines in certain populations such as newborns and pregnant women. Thus, the optimization of vaccination strategies that circumvent this limitation is a priority. This study evaluated the efficacy of a single dose subunit vaccine based on recombinant Hsp65 protein against infection with M. tuberculosis H37Rv. The Hsp65 protein in this study was either associated or not with immunostimulants, and was encapsulated in biodegradable PLGA microspheres. Our results demonstrate that the protein was entrapped in microspheres of adequate diameter to be engulfed by phagocytes. Mice vaccinated with a single dose of Hsp65-microspheres or Hsp65+CpG-microspheres developed both humoral and cellular-specific immune responses. However, they did not protect mice against challenge with M. tuberculosis. By contrast, Hsp65+KLK-microspheres induced specific immune responses that reduced bacilli loads and minimized lung parenchyma damage. These data suggest that a subunit vaccine based on recombinant protein Hsp65 is feasible.

HSP65 DNA as therapeutic strategy to treat experimental paracoccidioidomycosis.

The conventional treatment for paracoccidioidomycosis, the most prevalent mycosis in Latin America, involves long periods of therapy resulting in sequels and high frequency of relapses. The search for new alternatives of treatment is necessary. Previously, we have demonstrated that the hsp65 gene from Mycobacterium leprae shows prophylactic effects against murine paracoccidioidomycosis. Here, we tested the DNAhsp65 immunotherapy in BALB/c mice infected with Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis. We observed an increase of Th1 cytokines accompanied by a reduction in fungal burden and pulmonary injury. These results provide new prospects for immunotherapy of paracoccidioidomycosis and other mycoses.

Vaccination with a Sindbis virus-based DNA vaccine expressing antigen 85B induces protective immunity against Mycobacterium tuberculosis.

To improve DNA vaccination against Mycobacterium tuberculosis, we evaluated the effectiveness of a Sindbis virus-based DNA construct expressing the tuberculosis antigen 85B (Sin85B). The protective efficacy of Sin85B was initially assessed by aerogenically challenging immunized C57BL/6 mice with virulent Mycobacterium tuberculosis. At 1 and 7 months postinfection, the lung bacterial burdens were considerably reduced and the lung pathology was improved in vaccinated mice compared to naive controls. Furthermore, the mean survival period for Sin85B-immunized mice (305 +/- 9 days) after the tuberculous challenge was extended 102 days relative to the naive mice (203 +/- 13 days) and was essentially equivalent to the survival time of Mycobacterium bovis BCG-vaccinated mice (294 +/- 15 days). The essential role of gamma interferon (IFN-gamma) in Sin85B-mediated protection was established by showing that significantly increased levels of IFN-gamma mRNA were present postinfection in lung cells from vaccinated mice relative to control mice and by demonstrating that IFN-gamma depletion prior to challenge abolished the vaccine-induced protection. The substantial antituberculosis protective responses induced by Sin85B immunization of CD4-/- mice strongly suggested that CD8 cells partially mediate Sin85B-induced protective immunity. Interestingly, Sin85B vaccination did not protect RNase L-/- (a key enzyme in the innate antiviral response) mice while significant protection was detected in RNase L-/- mice immunized with either BCG or a conventional DNA plasmid expressing antigen 85B. These data show that immunization with Sin85B offers protection similar to BCG in a murine model of pulmonary tuberculosis and suggest that Sin85B-induced protection is dependent upon both innate and acquired immune mechanisms.

Factors associated with severe granulomatous pneumonia in Mycobacterium tuberculosis-infected mice vaccinated therapeutically with hsp65 DNA.

Resistant C57BL/6 mice infected in the lungs with Mycobacterium tuberculosis and then therapeutically vaccinated with Mycobacterium leprae-derived hsp65 DNA develop severe granulomatous pneumonia and tissue damage. Analysis of cells accumulating in the lungs of these animals revealed substantial increases in T cells secreting tumor necrosis factor alpha and CD8 cells staining positive for granzyme B. Stimulation of lung cells ex vivo revealed very high levels of interleukin-10, some of which was produced by B-1 B cells. This was probably an anti-inflammatory response, since lung pathology was dramatically worsened in B-cell gene-disrupted mice.

Designer arrays for defined mutant analysis to detect genes essential for survival of Mycobacterium tuberculosis in mouse lungs.

The mechanisms by which Mycobacterium tuberculosis elicits disease are complex, involving a large repertoire of bacterial genes that are required for in vivo growth and survival. To identify such genes, we utilized a high-throughput microarray detection method to rapidly screen hundreds of unique, genotypically defined transposon mutants for in vivo survival with a high degree of specificity and sensitivity. Thirty-one M. tuberculosis genes were found to be required for in vivo survival in mouse lungs. These genes are involved in a broad range of activities, including metabolism, cell wall functions, and regulation. Our screen included 11 of the 12 known members of the mycobacterial membrane protein (mmpL) family genes, and mutation of 6 of these genes-mmpL4, mmpL5, mmpL7, mmpL8, mmpL10, and mmpL11-severely compromised the ability of the mutants to multiply in mouse lungs. Most of the 31 genes are conserved in other pathogenic mycobacteria, including M. leprae and M. bovis, suggesting that a core of basic in vivo survival mechanisms may be highly conserved despite the divergent human pathology caused by members of the mycobacterial genus. Of the 31 genes reported here, 17 have not been previously described to be involved in in vivo growth and survival of M. tuberculosis.

Effect of microbial heat shock proteins on airway inflammation and hyperresponsiveness.

Microbial heat shock proteins (hsp) have been associated with the generation and induction of Th1-type immune responses. We tested the effects of treatment with five different microbial hsp (Mycobacterium leprae, Streptococcus pneumoniae, Helicobacter pylori, bacillus Calmette-Guérin, and Mycobacterium tuberculosis) in a murine model of allergic airway inflammation and airway hyperresponsiveness (AHR). Mice were sensitized to OVA by i.p. injection and then challenged by OVA inhalation. Hsp were administered to each group by i.p. injection before sensitization and challenge. Sensitized and challenged mice developed increased serum levels of OVA-specific IgE with significant airway eosinophilia and heightened responsiveness to methacholine when compared with nonsensitized animals. Administration of M. leprae hsp prevented both development of AHR as well as bronchoalveolar lavage fluid eosinophilia in a dose-dependent manner. Treatment with M. leprae hsp also resulted in suppression of IL-4 and IL-5 production in bronchoalveolar lavage fluid, while IL-10 and IFN-gamma production were increased. Furthermore, M. leprae hsp treatment significantly suppressed OVA-specific IgE production and goblet cell hyperplasia/mucin hyperproduction. In contrast, treatment with the other hsp failed to prevent changes in airway responsiveness, lung eosinophilia, or cytokine production. Depletion of gamma/delta T lymphocytes before sensitization and challenge abolished the effect of M. leprae hsp treatment on AHR. These results indicate selective and distinctive properties among the hsp, and that M. leprae hsp may have a potential therapeutic role in the treatment of allergic airway inflammation and altered airway function.

Isolation of cultivable mycobacteria from feces and lungs of armadillos infected with Mycobacterium leprae.

In the past, no cultivable mycobacteria were isolated from armadillos captured in the state of Florida, U.S.A. But recent findings of acid-fast bacilli (AFB) in the lungs of armadillos infected with Mycobacterium leprae prompted us to undertake this study to determine the correlation between systemic leprosy infection and the occurrence of cultivable mycobacteria in the lungs and stools of these animals. No AFB could be isolated from noninfected animals. Seventy percent of the infected animals developed disseminated infection, but no cultivable mycobacteria were isolated from their livers and spleens. However, cultivable mycobacteria were isolated from the lungs and stools of a large number of armadillos showing disseminated infection. The most common among these were M. gordonae, M. fortuitum, and M. avium. There was a close correlation between the development of disseminated leprosy infection and the occurrence of cultivable mycobacteria in their lungs and stools, perhaps due to the decline in the immune system in these animals in the later stages of infection.

Leprosy in wild armadillos (Dasypus novemcinctus) on the Texas Gulf Coast: anatomic pathology.

Recent studies of the "naturally occurring leprosy-like disease of wild armadillos" establish that the causative bacillus is genetically identical to M. leprae from human sources, and thus the disease is a zoonosis, sylvatic leprosy. A recent survey of 451 wild armadillos from the Texas Gulf Coast demonstrated sylvatic leprosy in 4.66%. This companion study reports the anatomic pathologic changes seen in the 17 leprous and 17 nonleprous armadillos necropsied in that survey. Findings support previous studies on the histopathology and pathogenesis of sylvatic leprosy, but a broader spectrum of histologic changes are noted.

Replication of Mycobacterium leprae in hibernating ground squirrels (Citellus tridecemlineatus).

Subcutaneous injections of 10(6) acid-fast bacteria from human lepromatous tissue to hibernating ground squirrels (Citellus tridecemlineatus) is followed by generalized infection of the animals and spontaneous death. The number of mycobacteria is high in the skin of GS during winter and decreases during summer. Animals surviving the first hibernation period invariably die during the second. The number of microorganism in the visceral organs is low at all times. The system of infected hibernating GS is a valuable model for the experimental study of leprosy.

Variably acid-fast bacteria in a case of systemic sarcoidosis and hypodermitis sclerodermiformis

The histologic finding of variably acid-fast coccoid forms in all the available biopsy material (skin, lymph nodes, and lung) from a case of coexisting scleroderma-like cutaneous disease (hypodermitis sclerodermiformis) and systemic sarcoidosis is reported. The morphologic size, shape, and staining characteristics of these microbes, along with the presence of the lung of 'large bodies', suggest that these microbes are cell wall deficient L forms of mycobacteria. Culture of the skin of the scleroderma-like lesion yielded Staphylococcus epidermidis, and the relationship of this isolate to the histologic findings of bacteria is discussed, as well as the possible pathogenic role played by L forms of mycobacteria in collagen disease and systemic sarcoidosis.