On-Slide Heat Sterilization Enables Mass Spectrometry Imaging of Tissue Infected with High-Threat Pathogens Outside of Biocontainment: A Study Directed at Mycobacterium tuberculosis.

Mass spectrometry imaging investigations of tissues infected with agents that require high-security biocontainment, such as Mycobacterium tuberculosis, have been limited due to incompatible sterilization techniques. Here we describe an on-slide heat sterilization method that enables mass spectrometry imaging investigations of pharmaceuticals, lipids, and metabolites in infected tissue samples outside of biocontainment. An evaluation of different temperatures and incubation times determined that 100 °C for 1 h was essential to sterilize 5 times the bacterial burden observed in tuberculosis (TB) cavity sections. Laser-capture microdissection combined with liquid chromatography with tandem mass spectrometry quantitation, in addition to mass spectrometry imaging, showed that no degradation was observed following the on-slide heat sterilization protocol for a variety of drug classes covering a range of physicochemical properties. Utilizing the tissue mimetic model, we demonstrated that the detection of lipid and metabolite ions was not impacted by heat sterilization and that, for several metabolites, the on-slide heat sterilization method improved the sensitivity when compared to control samples. An application of the on-slide heat sterilization to M. tuberculosis infected tissue enabled the first detection and spatial distribution of lipids indicative of a lysosomal storage disease phenotype within TB granuloma macrophages, in addition to the differential distribution of metabolites central to the fatty acid oxidation pathway. These initial investigations detected a pronounced heterogeneity within the cellular regions and necrotic cores of individual TB granulomas and across different evolving granulomas. This study provides the framework for mass spectrometry imaging investigations of high-threat pathogens outside of biocontainment.

Development of a novel self-sanitizing mask prototype to combat the spread of infectious disease and reduce unnecessary waste.

With the spread of COVID-19, significant emphasis has been placed on mitigation techniques such as mask wearing to slow infectious disease transmission. Widespread use of face coverings has revealed challenges such as mask contamination and waste, presenting an opportunity to improve the current technologies. In response, we have developed the Auto-sanitizing Retractable Mask Optimized for Reusability (ARMOR). ARMOR is a novel, reusable face covering that can be quickly disinfected using an array of ultraviolet C lamps contained within a wearable case. A nanomembrane UVC sensor was used to quantify the intensity of germicidal radiation at 18 different locations on the face covering and determine the necessary exposure time to inactivate SARS-CoV-2 in addition to other viruses and bacteria. After experimentation, it was found that ARMOR successfully provided germicidal radiation to all areas of the mask and will inactivate SARS-CoV-2 in approximately 180 s, H1N1 Influenza in 130 s, and Mycobacterium tuberculosis in 113 s, proving that this design is effective at eliminating a variety of pathogens and can serve as an alternative to traditional waste-producing disposable face masks. The accessibility, ease of use, and speed of sanitization supports the wide application of ARMOR in both clinical and public settings.

The intrinsic ATPase activity of Mycobacterium tuberculosis UvrC is crucial for its damage-specific DNA incision function.

To ensure genome stability, bacteria have evolved a network of DNA repair mechanisms; among them, the UvrABC-dependent nucleotide excision repair (NER) pathway is essential for the incision of a variety of bulky adducts generated by exogenous chemicals, UV radiation and by-products of cellular metabolism. However, very little is known about the enzymatic properties of Mycobacterium tuberculosis UvrABC excinuclease complex. Furthermore, the biochemical properties of Escherichia coli UvrC (EcUvrC) are not well understood (compared to UvrA and UvrB), perhaps due to its limited availability and/or activity instability in vitro. In addition, homology modelling of M. tuberculosis UvrC (MtUvrC) revealed the presence of a putative ATP-binding pocket, although its function remains unknown. To elucidate the biochemical properties of UvrC, we constructed and purified wild-type MtUvrC and its eight variants harbouring mutations within the ATP-binding pocket. The data from DNA-binding studies suggest that MtUvrC exhibits high-affinity for duplex DNA containing a bubble or fluorescein-dT moiety, over fluorescein-adducted single-stranded DNA. Most notably, MtUvrC has an intrinsic UvrB-independent ATPase activity, which drives dual incision of the damaged DNA strand. In contrast, EcUvrC is devoid of ATPase activity; however, it retains the ability to bind ATP at levels comparable to that of MtUvrC. The ATPase-deficient variants map to residues lining the MtUvrC ATP-binding pocket. Further analysis of these variants revealed separation of function between ATPase and DNA-binding activities in MtUvrC. Altogether, these findings reveal functional diversity of the bacterial NER machinery and a paradigm for the evolution of a catalytic scaffold in UvrC.

The efficacy of vacuum-ultraviolet light disinfection of some common environmental pathogens.

BACKGROUND: This study is to elucidate the disinfection effect of ozone producing low-pressure Hg vapor lamps against human pathogens. Ozone producing low-pressure Hg vapor lamps emit mainly 254 nm ultraviolet light C (UVC) with about 10% power of Vacuum-ultraviolet (VUV) light at 185 nm. The combination of UVC and VUV can inactivate airborne pathogens by disrupting the genetic materials or generation of reactive oxygen species, respectively. In this study, inactivation of common bacteria including Escherichia coli ATCC25922 (E. coli), Extended Spectrum Beta-Lactamase-producing E. coli (ESBL), Methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium tuberculosis (MTB), and that of influenza A viruses H1N1 and H3N2 under the radiation from ozone producing low-pressure Hg vapor lamps was examined. Log reduction values at different treatment durations were determined. METHODS: In vitro tests were carried out. Various bacterium and virus suspensions were added onto nitrocellulose filter papers and subjected to the illumination from ozone producing low-pressure Hg vapor lamps. The extents of pathogen inactivation at different illumination times were investigated by conducting a series of experiments with increasing duration of illumination. log10 reduction in CFU/ml and reduction at log10(TCID50) were respectively measured for bacteria and viruses. The disinfection effectiveness of this type of lamps against the pathogens under the environment with a moderate barrier to light was therefore evaluated. RESULTS: Ozone producing low-pressure Hg vapor lamp successfully inactivated these human pathogens. Nevertheless, among these pathogens, disinfection of MTB required more intense treatment. In the best tested situation, 3-log10 inactivation of pathogens can be achieved with ≤10 min of VUV treatment except MTB which needed about 20 min. This demonstrated the high resistance against UV disinfection of MTB. CONCLUSIONS: Following the criteria that valid germicidal results can be reflected with 3-log10 inactivation for bacteria, 4-log10 inactivation for viruses and 5-log10 inactivation for MTB, most of the bacteria required ≤10 min of VUV treatment, 20 min for the influenza viruses while MTB needed about 30 min VUV treatment. This indicated that VUV light is an effective approach against different environmental microorganisms.

Increased vitamin D receptor expression from macrophages after stimulation with M. tuberculosis among persons who have recovered from extrapulmonary tuberculosis.

BACKGROUND: Independent of HIV infection, extrapulmonary TB (EPTB) risk is increased in women, persons of black race or foreign birth, and by genetic variants in vitamin D receptor (VDR), interleukin-1 beta (IL-1ß), and toll-like receptor (TLR)-2; functional correlates are unclear. We evaluated macrophage expression of VDR, TLR2, cathelicidin, and TNF-α, and production of IL-1ß in HIV-seronegative persons with previous EPTB, previous pulmonary TB, latent M. tuberculosis infection, and uninfected TB contacts. Persons with previous pleural TB were excluded due to enhanced immune responses at the site of disease. METHODS: Macrophages were stimulated with TLR-2 agonist M. tuberculosis lipoprotein (LpqH), live and gamma-irradiated M. tuberculosis. RESULTS: M. tuberculosis - infected macrophages from persons with previous EPTB had increased VDR expression (29.17 relative value unit increase in median expression vs. uninfected contacts, after adjusting for foreign-born status; P = 0.02). Macrophages from persons with previous EPTB had a 38.88 µg/mL increase in median IL-1ß production after stimulation with LpqH compared to uninfected contacts (P = 0.01); the effect was similar (44.99 µg/mL) but not statistically significant after controlling for foreign-born status. Median 25-hydroxyvitamin D levels were low but not significantly different between groups. CONCLUSIONS: There was increased macrophage expression of VDR after stimulation with live M. tuberculosis in persons with previous extrapulmonary TB. If post-treatment VDR expression reflects expression prior to disease, it may identify persons at risk for extrapulmonary TB.

Mycobacterium tuberculosis-specific CD4+ and CD8+ T cells differ in their capacity to recognize infected macrophages.

Containment of Mycobacterium tuberculosis (Mtb) infection requires T cell recognition of infected macrophages. Mtb has evolved to tolerate, evade, and subvert host immunity. Despite a vigorous and sustained CD8+ T cell response during Mtb infection, CD8+ T cells make limited contribution to protection. Here, we ask whether the ability of Mtb-specific T cells to restrict Mtb growth is related to their capacity to recognize Mtb-infected macrophages. We derived CD8+ T cell lines that recognized the Mtb immunodominant epitope TB10.44-11 and compared them to CD4+ T cell lines that recognized Ag85b240-254 or ESAT63-17. While the CD4+ T cells recognized Mtb-infected macrophages and inhibited Mtb growth in vitro, the TB10.4-specific CD8+ T cells neither recognized Mtb-infected macrophages nor restricted Mtb growth. TB10.4-specific CD8+ T cells recognized macrophages infected with Listeria monocytogenes expressing TB10.4. However, over-expression of TB10.4 in Mtb did not confer recognition by TB10.4-specific CD8+ T cells. CD8+ T cells recognized macrophages pulsed with irradiated Mtb, indicating that macrophages can efficiently cross-present the TB10.4 protein and raising the possibility that viable bacilli might suppress cross-presentation. Importantly, polyclonal CD8+ T cells specific for Mtb antigens other than TB10.4 recognized Mtb-infected macrophages in a MHC-restricted manner. As TB10.4 elicits a dominant CD8+ T cell response that poorly recognizes Mtb-infected macrophages, we propose that TB10.4 acts as a decoy antigen. Moreover, it appears that this response overshadows subdominant CD8+ T cell response that can recognize Mtb-infected macrophages. The ability of Mtb to subvert the CD8+ T cell response may explain why CD8+ T cells make a disproportionately small contribution to host defense compared to CD4+ T cells. The selection of Mtb antigens for vaccines has focused on antigens that generate immunodominant responses. We propose that establishing whether vaccine-elicited, Mtb-specific T cells recognize Mtb-infected macrophages could be a useful criterion for preclinical vaccine development.

Mapeamento e distribuição dos isolados do complexo mycobacterium tuberculosis provenientes de casos de tuberculose bovino em Moçambique / Mapping and distribution of mycobacterium tuberculosis complex isolates from bovine tuberculosis cases in Mozambique

A tuberculose bovina (bTB) é um problema sanitário importante em Moçambique e ainda a espera de uma ação organizada por parte dos Serviços Veterinários Oficiais. O presente estudo teve por objetivo investigar e mapear os genótipos de Mycobacterium bovis circulantes no país e paralelamente maximizar a utilização de abatedouros como fonte de informação epidemiológica da bTB. Durante o período de outubro de 2016 a abril de 2017 foram colhidas um total de 90 amostras de lesões sugestivas de tuberculose bovina nos abatedouros Municipais de Maputo e Maxixe e dois abatedouros privados da província de Maputo. Essas amostras, juntamente com outras 10, disponibilizadas pelo Laboratório Central de Veterinária e 72 do banco de amostras de Faculdade de Veterinária de Moçambique foram processadas para isolamento, identificação e discriminação molecular (MIRU-VNTR e spoligotyping) de micobactérias. Nos abatedouros foram coletados dados para calcular as prevalências de carcaças com lesões sugestivas de tuberculose e foi estimada em 0,63% e 80% das carcaças condenadas por tuberculose apresentaram lesões compatíveis com generalização da infecção. Foram obtidos 104 isolados identificados como gênero Mycobacterium, dos quais 80 foram compatíveis com o MTBC e 10 MNT. Destes 80 MTBC, 70 foram identificados como M. bovis. O MIRU-VNTR discriminou os 70 isolados de M. bovis em 47 perfis, agrupados em 3 complexos clonais e cinco singletons. Dos 24 loci estudados, os que apresentaram maiores polimorfismos foram MIRU 960, 2996 e QUB-4052. Em relação ao spoligotyping, foram identificados cinco perfis, dos quais o mais prevalente foi o SB0961, seguido do SB0140, SB2306, SB2481 (novo) e SB1099. Dos 70 isolados submetidos a análises dos complexos clonais africano 1, africano 2, europeu 1 e europeu 2, foram detectados apenas 18,5% de europeu. O distrito de Machanga foi o que apresentou maior diversidade de isolados e o Govuro maior número de isolados de M. bovis. Quando comparados as técnicas, o MIRU-VNTR apresentou maior poder de discriminação em relação ao spoligotyping. O complexo clonal europeu 1 está relacionado com o SB0140 que por sua vez é característico de isolados do Reino Unido e de países que tiveram trocas comerciais de bovinos com o país, incluindo os circunvizinhos a Moçambique e de onde há registros da importação de animais para Moçambique. A não identificação precisa dos complexos clonais dos spoligotyping SB0961, SB2306, SB2481 relacionados, podem ser derivados do BCG, que é sugestivo de evolução clonal própria de Moçambique e os complexos clonais até hoje existentes não são suficientes para discriminar os isolados de Moçambique. Embora os dados de abatedouros sugeriram que a prevalência da tuberculose bovina em Moçambique está entre as mais baixas dos países africanos, a predominância de carcaças com lesões generalizadas significa alto risco de exposição de animais e humanos, sobretudo das populações rurais que têm estreito contato com esses animais. Esse risco é ampliado em função da alta prevalência de humanos que vivem com HIV/AIDS no país. Assim, recomenda-se que Moçambique estruture programa de controle da doença nos animais e métodos de diagnóstico que detectem a infecção por M. bovis na população humana

Bovine tuberculosis (bTB) is a major sanitary problem in Mozambique and awaits organized action by the Official Veterinary Services. The aim of this work was to investigate and map the circulating Mycobacterium bovis genotypes in the country and to maximize the use of slaughterhouses as a source of epidemiological information for bTB. During the period from October 2016 to April 2017, a total of 90 samples with lesions suggestive of bovine tuberculosis were collected from Maputo and Maxixe Municipal abattoirs and two private abattoirs from the province of Maputo. These samples, together with 10 others provided by the Central Veterinary Laboratory and 72 from the Veterinary Faculty sample bank were processed for isolation, identification and molecular discrimination (MIRU-VNTR and spoligotyping) of mycobacteria. Samples collected in the slaughterhouses were analyzed by calculating the prevalence of carcasses with lesions suggestive of bTB, which was estimated at 0.63% and 80% of carcasses condemned for tuberculosis presented lesions compatible with generalized infection. A total of 104 isolates were identified as M. bovis as Mycobacterium genus were obtained, of which 80 were compatible with MTBC and 10 MNT. Of these 80 MTBC, 70 were identified as M. bovis. The MIRU-VNTR discriminated the 70 isolates of M. bovis in 47 profiles, grouped in 3 clonal complexes and 5 singletons. Of the 24 loci studied, the ones with the highest polymorphisms were MIRU 960, 2996 and QUB-4052. In relation to spoligotyping, five profiles were identified; SB0961 was the most prevalent, being SB0961, followed by SB0140, SB2306, SB2481 (new) and SB1099. Of the 70 isolates submitted to analyzes of the clonal complexes African 1, African 2, European 1 and European 2, only 18.5% of European complex were detected. Machanga district presented the greatest diversity of isolates, while Govuro district had largest number of isolates of M. bovis. The MIRU-VNTR presented greater power of discrimination in relation to spoligotyping. The European clonal complex 1 was related to SB0140 which in turn is characteristic of isolates from the United Kingdom and from countries that have had commercial trade in cattle with UK including those surrounding Mozambique and where there are records of imports of animals for Mozambique. The precise identification of the clonal complexes of the SB0961, SB2306 and SB2481 related spoligotypings subject to the BCG derivatives, is suggestive of the clonal evolution of Mozambique and that the clonal complexes to date are not sufficient to discriminate against the isolates from Mozambique. Although data from slaughterhouses suggested that the prevalence of bovine tuberculosis in Mozambique is among the lowest in African countries, the predominance of carcasses with generalized lesions means a high risk of animal and human exposure, especially of rural populations that have close contact with these animals. This risk is amplified due to the high prevalence of people that living with HIV/AIDS in the country. Thus, it is recommended that Mozambique structure a disease control program in animals and diagnostic methods that detect M. bovis infection in the human population

Thermal lysis and isothermal amplification of Mycobacterium tuberculosis H37Rv in one tube.

Tuberculosis (TB) is a leading cause of high mortality rates in developing countries. Sample preparation is one of the major challenges in developing an inexpensive point-of-care device for rapid and confirmed detection of tuberculosis. Existing chemical and mechanical lysis methods are unsuitable for field applications, as they require intermediate wash steps, manual intervention or separate lysis equipment. We report a one-step reaction protocol (65°C and 60min) for the H37Rv strain of Mycobacterium tuberculosis that (i) completely disinfects the mycobacteria culture, (ii) lyses the cells and (iii) performs helicase dependent amplification on the extracted DNA. Our assay combines multiple functions in a single step, uses a dry heat bath and does not require any intermediate user intervention, which makes it suitable for use by minimally trained health workers at the point of care.

Primary macrophages and J774 cells respond differently to infection with Mycobacterium tuberculosis.

Macrophages play an essential role in the early immune response to Mycobacterium tuberculosis and are the cell type preferentially infected in vivo. Primary macrophages and macrophage-like cell lines are commonly used as infection models, although the physiological relevance of cell lines, particularly for host-pathogen interaction studies, is debatable. Here we use high-throughput RNA-sequencing to analyse transcriptome dynamics of two macrophage models in response to M. tuberculosis infection. Specifically, we study the early response of bone marrow-derived mouse macrophages and cell line J774 to infection with live and γ-irradiated (killed) M. tuberculosis. We show that infection with live bacilli specifically alters the expression of host genes such as Rsad2, Ifit1/2/3 and Rig-I, whose potential roles in resistance to M. tuberculosis infection have not yet been investigated. In addition, the response of primary macrophages is faster and more intense than that of J774 cells in terms of number of differentially expressed genes and magnitude of induction/repression. Our results point to potentially novel processes leading to immune containment early during M. tuberculosis infection, and support the idea that important differences exist between primary macrophages and cell lines, which should be taken into account when choosing a macrophage model to study host-pathogen interactions.

Infecciones bacterianas agudas del sistema nervioso central: un punto de vista radiológico / Acute bacterial infections of central nervous system: Radiological key points

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Mycobacterium tuberculosis RuvX is a Holliday junction resolvase formed by dimerisation of the monomeric YqgF nuclease domain.

The Mycobacterium tuberculosis genome possesses homologues of the ruvC and yqgF genes that encode putative Holliday junction (HJ) resolvases. However, their gene expression profiles and enzymatic properties have not been experimentally defined. Here we report that expression of ruvC and yqgF is induced in response to DNA damage. Protein-DNA interaction assays with purified M. tuberculosis RuvC (MtRuvC) and YqgF (MtRuvX) revealed that both associate preferentially with HJ DNA, albeit with differing affinities. Although both MtRuvC and MtRuvX cleaved HJ DNA in vitro, the latter displayed robust HJ resolution activity by symmetrically related, paired incisions. MtRuvX showed a higher binding affinity for the HJ structure over other branched recombination and replication intermediates. An MtRuvX(D28N) mutation, eliminating one of the highly conserved catalytic residues in this class of endonucleases, dramatically reduced its ability to cleave HJ DNA. Furthermore, a unique cysteine (C38) fulfils a crucial role in HJ cleavage, consistent with disulfide-bond mediated dimerization being essential for MtRuvX activity. In contrast, E. coli YqgF is monomeric and exhibits no branched DNA binding or cleavage activity. These results fit with a functional modification of YqgF in M. tuberculosis so that it can act as a dimeric HJ resolvase analogous to that of RuvC.

A transcriptional co-repressor regulatory circuit controlling the heat-shock response of Mycobacterium tuberculosis.

The co-ordinated regulation of heat shock proteins is critically important for the stress response of M. tuberculosis, failure of which results in enhanced immune recognition of the tubercle bacilli with reduced survival during chronic infections. In this study, we show that PhoP regulates the transcription of α-crystallin 2 (acr2), expression of which increases more than any other gene of M. tuberculosis during heat-shock or following macrophage infection. We also show that regulation of acr2 by PhoP is attributable to direct regulator-promoter interactions at specific sites proximal to a sequence motif comprising the target site of another virulence factor, HspR. While both these regulators, on their own, are capable of influencing acr2 expression, remarkably our results show that the two virulence regulators PhoP and HspR interact with each other to influence their in vivo recruitment at the acr2 regulatory region, and in turn, contribute to stress-specific regulation of acr2 expression. We propose a model to suggest how protein-protein interactions between PhoP and HspR influence the regulation of α-crystallin 2, an essential pathogenic determinant of M. tuberculosis.

Either non-homologous ends joining or homologous recombination is required to repair double-strand breaks in the genome of macrophage-internalized Mycobacterium tuberculosis.

The intracellular pathogen Mycobacterium tuberculosis (Mtb) is constantly exposed to a multitude of hostile conditions and is confronted by a variety of potentially DNA-damaging assaults in vivo, primarily from host-generated antimicrobial toxic radicals. Exposure to reactive nitrogen species and/or reactive oxygen species causes different types of DNA damage, including oxidation, depurination, methylation and deamination, that can result in single- or double-strand breaks (DSBs). These breaks affect the integrity of the whole genome and, when left unrepaired, can lead to cell death. Here, we investigated the role of the DSB repair pathways, homologous recombination (HR) and non-homologous ends joining (NHEJ), in the survival of Mtb inside macrophages. To this end, we constructed Mtb strains defective for HR (ΔrecA), NHEJ [Δ(ku,ligD)], or both DSB repair systems [Δ(ku,ligD,recA)]. Experiments using these strains revealed that either HR or NHEJ is sufficient for the survival and propagation of tubercle bacilli inside macrophages. Inhibition of nitric oxide or superoxide anion production with L-NIL or apocynin, respectively, enabled the Δ(ku,ligD,recA) mutant strain lacking both systems to survive intracellularly. Complementation of the Δ(ku,ligD,recA) mutant with an intact recA or ku-ligD rescued the ability of Mtb to propagate inside macrophages.

Inactivation of multidrug resistant (MDR)- and extensively drug resistant (XDR)-Mycobacterium tuberculosis by photodynamic therapy.

We investigated the effects of photodynamic therapy (PDT) on anti-tuberculosis (TB) activity by measuring inactivation rates, expressed as D-value, of MDR- and XDR-Mycobacterium tuberculosis (M. tb) clinical strains in vitro. Approximately 10(6) colony forming unit per milliliter (CFU/ml) of the bacilli were irradiated with various doses of laser light after exposure to photosensitizers. Survival of M. tb was measured by enumerating CFU in 7H10 medium to measure D-values. No inactivation of M. tb was observed when exposed to photosensitizers (radachlorin or DH-I-180-3) only or laser light only (P>0.1). Treatment with a combination of photosentizer and laser inactivated M. tb although there was a significant difference between the types of photosensitizers applied (P<0.05). Linear inactivation curves for the clinical M. tb strains were obtained up to laser doses of 30 J/cm(2) but prolonged irradiation did not linearly inactivate M. tb, yielding sigmoid PDT inactivation curves. D-values of M. tb determined from the slope of linear regression lines in PDT were not significantly different and ranged from 10.50 to 12.13 J/cm(2) with 670 nm laser irradiation at 100 mW/cm(2) of the fluency rate, except for a drug-susceptible strain among the clinical strains tested. This suggests that PDT inactivated M. tb clinical strains regardless of drug resistance levels of the bacilli. Intermittent and repeated PDT allowed acceleration of the inactivation of the bacilli as a way to avoid the sigmoid inactivation curves. In conclusion, PDT could be alternative as a new option for treatment for MDR- and XDR-tuberculosis.

The role of lipid raft aggregation in the infection of type II pneumocytes by Mycobacterium tuberculosis.

Dynamic, cholesterol-dense regions of the plasma membrane, known as lipid rafts (LR), have been observed to develop during and may be directly involved in infection of host cells by various pathogens. This study focuses on LR aggregation induced in alveolar epithelial cells during infection with Mycobacterium tuberculosis (Mtb) bacilli. We report dose- and time-dependent increases in LR aggregation after infection with three different strains at multiplicities of infection of 1, 10 and 100 from 2-24 hr post infection (hpi). Specific strain-dependent variations were noted among H37Rv, HN878 and CDC1551 with H37Rv producing the most significant increase from 15 aggregates per cell (APC) to 27 APC at MOI 100 during the 24 hour infection period. Treatment of epithelial cells with Culture Filtrate Protein, Total Lipids and gamma-irradiated whole cells from each strain failed to induce the level of LR aggregation observed during infection with any of the live strains. However, filtered supernatants from infected epithelial cells did produce comparable LR aggregation, suggesting a secreted mycobacterial product produced during infection of host cells is responsible for LR aggregation. Disruption of lipid raft formation prior to infection indicates that Mtb bacilli utilize LR aggregates for internalization and survival in epithelial cells. Treatment of host cells with the LR-disruption agent Filipin III produced a nearly 22% reduction in viable bacteria for strains H37Rv and HN878, and a 7% reduction for strain CDC1551 after 6 hpi. This study provides evidence for significant mycobacterial-induced changes in the plasma membrane of alveolar epithelial cells and that Mtb strains vary in their ability to facilitate aggregation and utilization of LR.

Mycobacterium tuberculosis uvrC essentiality in response to UV-induced cell damage.

Control of tuberculosis depends both on an effective, accurate, and rapid diagnosis and an effective treatment and management. Antituberculous drugs have been used for more than 50 years and are likely ineffective against multidrug-resistant strains, leading to an urgent need for new drugs. Comparative genome analysis has indicated that Mycobacterium tuberculosis uvrC, a component of nucleotide excision repair (NER) system, is an essential gene without any human homolog. This raises the possibility to use this gene as a new drug target. This study investigated the essential role of uvrC on growth of M. tuberculosis in the presence of DNA damaging agents, UV light and hydrogen peroxide (generator of reactive oxygen species). Results revealed that the M. tuberculosis uvrC mutant was more sensitive to UV than the control strain (p < 0.01), but was not more sensitive to hydrogen peroxide. These results showed that uvrC is essential for M. tuberculosis DNA repair system, particularly in response to DNA damage caused by UV irradiation.

Cryopreservation of Mycobacterium tuberculosis complex cells.

Successful long-term preservation of Mycobacterium tuberculosis cells is important for sample transport, research, biobanking, and the development of new drugs, vaccines, biomarkers, and diagnostics. In this report, Mycobacterium bovis bacillus Calmette-Guérin and M. tuberculosis H37Ra were used as models of M. tuberculosis complex strains to study cryopreservation of M. tuberculosis complex cells in diverse sample matrices at different cooling rates. Cells were cryopreserved in diverse sample matrices, namely, phosphate-buffered saline (PBS), Middlebrook 7H9 medium with or without added glycerol, and human sputum. The efficacy of cryopreservation was quantified by microbiological culture and microscopy with BacLight LIVE/DEAD staining. In all sample matrices examined, the microbiological culture results showed that the cooling rate was the most critical factor influencing cell viability. Slow cooling (a few degrees Celsius per minute) resulted in much higher M. tuberculosis complex recovery rates than rapid cooling (direct immersion in liquid nitrogen) (P < 0.05). Among the three defined cryopreservation media (PBS, 7H9, and 7H9 plus glycerol), there was no significant differential effect on viability (P = 0.06 to 0.87). Preincubation of thawed M. tuberculosis complex cells in 7H9 broth for 20 h before culture on solid Middlebrook 7H10 plates did not help the recovery of the cells from cryoinjury (P = 0.14 to 0.71). The BacLight LIVE/DEAD staining kit, based on Syto 9 and propidium iodide (PI), was also applied to assess cell envelope integrity after cryopreservation. Using the kit, similar percentages of "live" cells with intact envelopes were observed for samples cryopreserved under different conditions, which was inconsistent with the microbiological culture results. This implies that suboptimal cryopreservation might not cause severe damage to the cell wall and/or membrane but instead cause intracellular injury, which leads to the loss of cell viability.

Bactericidal effects of 405 nm light exposure demonstrated by inactivation of Escherichia, Salmonella, Shigella, Listeria, and Mycobacterium species in liquid suspensions and on exposed surfaces.

The bactericidal effect of 405 nm light was investigated on taxonomically diverse bacterial pathogens from the genera Salmonella, Shigella, Escherichia, Listeria, and Mycobacterium. High-intensity 405 nm light, generated from an array of 405-nm light-emitting diodes (LEDs), was used to inactivate bacteria in liquid suspension and on exposed surfaces. L. monocytogenes was most readily inactivated in suspension, whereas S. enterica was most resistant. In surface exposure tests, L. monocytogenes was more susceptible than Gram-negative enteric bacteria to 405 nm light when exposed on an agar surface but interestingly less susceptible than S. enterica after drying onto PVC and acrylic surfaces. The study findings, that 405 nm light inactivates diverse types of bacteria in liquids and on surfaces, in addition to the safety advantages of this visible (non-UV wavelength) light, indicate the potential of this technology for a range of decontamination applications.

Identification of a chemical that inhibits the mycobacterial UvrABC complex in nucleotide excision repair.

Bacterial DNA can be damaged by reactive nitrogen and oxygen intermediates (RNI and ROI) generated by host immunity, as well as by antibiotics that trigger bacterial production of ROI. Thus a pathogen's ability to repair its DNA may be important for persistent infection. A prominent role for nucleotide excision repair (NER) in disease caused by Mycobacterium tuberculosis (Mtb) was suggested by attenuation of uvrB-deficient Mtb in mice. However, it was unknown if Mtb's Uvr proteins could execute NER. Here we report that recombinant UvrA, UvrB, and UvrC from Mtb collectively bound and cleaved plasmid DNA exposed to ultraviolet (UV) irradiation or peroxynitrite. We used the DNA incision assay to test the mechanism of action of compounds identified in a high-throughput screen for their ability to delay recovery of M. smegmatis from UV irradiation. 2-(5-Amino-1,3,4-thiadiazol-2-ylbenzo[f]chromen-3-one) (ATBC) but not several closely related compounds inhibited cleavage of damaged DNA by UvrA, UvrB, and UvrC without intercalating in DNA and impaired recovery of M. smegmatis from UV irradiation. ATBC did not affect bacterial growth in the absence of UV exposure, nor did it exacerbate the growth defect of UV-irradiated mycobacteria that lacked uvrB. Thus, ATBC appears to be a cell-penetrant, selective inhibitor of mycobacterial NER. Chemical inhibitors of NER may facilitate studies of the role of NER in prokaryotic pathobiology.

Mycobacterium tuberculosis MT2816 encodes a key stress-response regulator.

BACKGROUND: Sigma (sigma) factors are transcription initiation factors that modulate the response of Mycobacterium tuberculosis to changes in extracellular milieu, allowing it to survive stress. METHODS: We analyzed the expression of MT2816/Rv2745c under various stress conditions that mimic the intracellular environment faced by M. tuberculosis. RESULTS: MT2816/Rv2745c expression was induced in M. tuberculosis following redox stress, heat shock and acid shock and intracellular replication. Its expression was also induced by SDS and thioridazine, agents that impact M. tuberculosis cell-envelope. However, exposure to isoniazid or ethambutol, front-line antituberculosis drugs which also target the cell envelope, did not induce the expression of MT2816/Rv2745c. Studies using Delta-sigma(H) and Delta-sigma(E) mutants showed that sigma(H) was required for the induction of MT2816/Rv2745c. Conditional expression of the MT2816/Rv2745c in M. tuberculosis showed that apart from regulating proteolysis, this gene may control the expression of trehalose biosynthesis and impact the maintenance of cellular redox potential and energy generation. CONCLUSIONS: The protein encoded by MT2816/Rv2745c is important for the pathogen's response to stress conditions that mimic in vivo growth and it is subject to complex regulation. The MT2816/Rv2745c encoded protein likely functions by protecting intracellular redox potential and by inducing the expression of trehalose, a constituent of M. tuberculosis cell walls that is important for defense against cell-surface and oxidative stress.