Sequence similarity of HSP65 of Mycobacterium bovis BCG with SARS-CoV-2 spike and nuclear proteins: may it predict an antigen-dependent immune protection of BCG against COVID-19?

The Bacillus Calmette-Guérin (BCG) vaccine is known to have protective effects not only against tuberculosis but also against other unrelated infectious diseases caused by different pathogens. Several epidemiological studies have also documented the beneficial influence of BCG vaccine in reducing both susceptibility to and severity of SARS-CoV-2 infection. The protective, non-specific effects of BCG vaccination would be related to an antigen-independent enhancement of the innate immunity, termed trained immunity. However, the knowledge that heat shock protein (HSP)65 is the main antigen of Mycobacterium bovis BCG prompted us to verify whether sequence similarity existed between HSP65 and SARS-CoV-2 spike (S) and nuclear (N) proteins that could support an antigen-driven immune protection of BCG vaccine. The results of the in silico investigation showed an extensive sequence similarity of HSP65 with both the viral proteins, especially SARS-CoV-2 S, that also involved the regions comprising immunodominant epitopes. The finding that the predicted B cell and CD4+ T cell epitopes of HSP65 shared strong similarity with the predicted B and T cell epitopes of both SARS-CoV-2 S and N would support the possibility of a cross-immune reaction of HSP65 of BCG with SARS-CoV-2.

A mycobacteriophage genomics approach to identify novel mycobacteriophage proteins with mycobactericidal properties.

Mycobacteriophages that are specific to mycobacteria are sources of various effector proteins that are capable of eliciting bactericidal responses. We describe a genomics approach in combination with bioinformatics to identify mycobacteriophage proteins that are toxic to mycobacteria upon expression. A genomic library comprising phage genome collections was screened for clones capable of killing Mycobacterium smegmatis strain mc2155. We identified four unique clones: clones 45 and 12N (from the mycobacteriophage D29) and clones 66 and 85 (from the mycobacteriophage Che12). The gene products from clones 66 and 45 were identified as Gp49 of the Che12 phage and Gp34 of the D29 phage, respectively. The gene products of the other two clones, 85 and 12N, utilized novel open reading frames (ORFs) coding for synthetic proteins. These four clones (clones 45, 66, 85 and 12N) caused growth defects in M. smegmatis and Mycobacterium bovis upon expression. Clones with Gp49 and Gp34 also induced growth defects in Escherichia coli, indicating that they target conserved host machineries. Their expression induced various morphological changes, indicating that they affected DNA replication and cell division steps. We predicted that Gp34 is a Xis protein that is required in phage DNA excision from the bacterial chromosome. Gp49 is predicted to have an HTH motif with DNA-bending/twisting properties. We suggest that this methodology is useful to identify new phage proteins with the desired properties without laboriously characterizing the individual phages. It is universal and could be applied to other bacteria-phage systems. We speculate that the existence of a virtually unlimited number of phages with unique gene products could offer a cheaper and less hazardous alternative to explore new antimicrobial molecules.

The Mycobacterium tuberculosis CRISPR-Associated Cas1 Involves Persistence and Tolerance to Anti-Tubercular Drugs.

Tuberculosis remains one of the leading causes of death worldwide. Even if new antitubercular drugs are currently being developed, the rapid emergence and spread of drug-resistant strain remain a severe challenge. The CRISPR associated proteins 1 (Cas1), a most conserved endonuclease which is responsible for spacer integration into CRISPR arrays, was found deleted in many specific drug-resistant strains. The function of Cas1 is still unknown in Mycobacterium type III-A CRISPR family. In this study, the Cas1 (Rv2817c) defect was found in 57.14% of clinical isolates. To investigate the function of Cas1 in new spacer acquisition, we challenged Bacillus Calmette-Guérin (BCG) with a mycobacteriophage D29. Newly acquired spacer sequence matches D29 genome was not found by spacer deep-sequencing. We further expressed Cas1 in recombinant Mycobacterium smegmatis. We found that Cas1 increased the sensitivity to multiple anti-tuberculosis drugs by reducing the persistence during drug treatment. We also showed that Cas1 impaired the repair of DNA damage and changed the stress response of Mycobacterium smegmatis. This study provides a further understanding of Cas1 in Mycobacterium tuberculosis complex (MTBC) drug-resistance evolution and a new sight for the tuberculosis treatment.

Mosaic structure of Mycobacterium bovis BCG genomes as a representation of phage sequences' mobility.

BACKGROUND: The control of genome stability is relevant for the worldwide BCG vaccine preventing the acute forms of childhood tuberculosis. BCG sub-strains whole genome comparative analysis and revealing the triggers of sub-strains transition were the purpose of our investigation. RESULTS: Whole genome sequencing of three BCG Russia seed lots (1963, 1982, 2006 years) confirmed the stability of vaccine sub-strain genome. Comparative analysis of three Mycobacteruim bovis and nine M. bovis BCG genomes shown that differences between "early" and "late" sub-strains BCG genomes were associated with specific prophage profiles. Several prophages common to all BCG genomes included ORFs which were homologues to Caudovirales. Surprisingly very different prophage profiles characterized BCG Tice and BCG Montreal genomes. These prophages contained ORFs which were homologues to Herpesviruses. Phylogeny of strains cohort based on genome maps restriction analysis and whole genomes sequence data were in agreement with prophage profiles. Pair-wise alignment of unique BCG Tice and BCG Montreal prophage sequences and BCG Russia 368 genome demonstrated only similarity of fragmetary sequences that suggested the contribution of prophages in genome mosaic structure formation. CONCLUSIONS: Control of the extended sequences is important for genome with mosaic structure. Prophage search tools are effective instruments in this analysis.

Tuberculosis generalizada en un cordero / Generalized tuberculosis in a lamb

Se describe un caso de tuberculosis generalizada en un cordero de tres meses de edad. Las lesiones sospechosas de tuberculosis fueron detectadas en los linfonodos traqueobronquial, mediastínico y hepático durante la inspección en matadero. La infección fue confirmada por histopatología, inmunohistoquímica y análisis molecular (AU)

The present report describes a case of tuberculosis in a 3 months-old lamb. Suspected lesions of tuberculosis in the lungs, tracheobronchial, mediastinal and hepatic lymph nodes were detected during abattoir meat inspection. The infection was confirmed by histopathology, immunohistochemistry and molecular analysis (AU)

Development of a bacteriophage phage replication assay for diagnosis of pulmonary tuberculosis.

Successful infection and replication of bacteriophages is indicative of the presence of viable bacteria. We describe here the development of a bacteriophage replication assay for the detection of Mycobacterium tuberculosis by using mycobacteriophage D29. Optimization of phage inoculate and incubation times allowed highly sensitive detection of M. bovis BCG. Fewer than 10 CFU (100 CFU/ml) were detected. No false-positive results were observed in negative samples. Application of the assay to 496 sputum specimens in the National Reference Laboratory of Zambia produced sensitivity, specificity, and positive and negative predictive values of 44.1, 92.6, 82.2, and 67.5%, respectively, compared to culture on Lowenstein-Jensen medium. The equivalent corresponding results for direct fluorescent smear microscopy were 42.3, 96.8, 91.2, and 67.6%. The small increase in sensitivity over that of direct microscopy does not justify the introduction of this technique for routine diagnosis of pulmonary tuberculosis at this time.

Plasmidic versus insertional cloning of heterologous genes in Mycobacterium bovis BCG: impact on in vivo antigen persistence and immune responses.

Bivalent recombinant strains of Mycobacterium bovis BCG (rBCG) expressing the early regulatory nef and the structural gag(p26) genes from the simian immunodeficiency virus (SIV) SIVmac251 were engineered so that both genes were cotranscribed from a synthetic operon. The expression cassette was cloned into a multicopy-replicating vector, and the expression levels of both nef and gag in the bivalent rBCG(nef-gag) strain were found to be comparable to those of monovalent rBCG(nef) or rBCG(gag) strains. However, extrachromosomal cloning of the nef-gag operon into a replicative plasmid resulted in strains of low genetic stability that rapidly lost the plasmid in vivo. Thus, the nef-gag operon was inserted site specifically into the BCG chromosome by means of mycobacteriophage Ms6-derived vectors. The resulting integrative rBCG(nef-gag) strains showed very high genetic stability both in vitro and in vivo. The in vivo expression of the heterologous genes was much longer lived when the expression cassette was inserted into the BCG chromosome. In one of the strains obtained, integrative cloning did not reduce the expression levels of the genes even though a single copy was present. Accordingly, this strain induced cellular immune responses of the same magnitude as that of the replicative rBCG strain containing several copies of the genes.

Avaliação do papel do interferon-y na modulação da resposta tecidual durante a infecção pelo Mycobacterium bovis / Evaluation of the role of interferon-y in the modulation of tissue response during infection with Mycobacterium bovis

O IFN-y tem sido implicado na defesa do hospedeiro contra as micobactérias. Esta citocina, produzida e liberada por linfócitos T recrutados para o sítio da infecção, é considerada o agente chave da ativação endógena, promovendo efeitos antimicobacterianos de macrófagos. Diversos modelos experimentais têm sido desenvolvidos para delinear o papel de citocinas responsáveis pelo controle da tuberculose. O presente estudo visa delinear o papel do IFN-y na modulação da resposta inflamatória granulomatosa no tecido hepático e pulmonar em camundongos infectados experimentalmente com o M. bovis. Os experimentos foram realizados utilizando-se camundongos com deficiência do gene responsável pela produção do IFN-y (C57BL/6 IFN-y-l-) e camundongos que produzem IFN-y (C57BL/6 IFN-y+l+), ambos infectados por via intravenosa com M. bovis. Avaliou-se a capacidade de sobrevida e multiplicação do M. bovis no fígado e pulmões, assim como características histopatógicas nestes dois órgãos. No curso da infecção (15, 30, 50 e 100 dias pós-infecção) a CFU (unidade formadora de colônia) foi quantificada no tecido hepático e pulmonar dos dois grupos de camundongos. A carga bacilar dos animais IFN-y-l- foi significantemente maior que nos IFN-y+/+. A quantidade de CFU foi maior no fígado que nos pulmões dos animais IFN-y-l- durante todo o estudo, enquanto nos IFN-y+/+ apenas no primeiro ponto estudado (15 dias pós-infecção). Adicionalmente, nos camundongos IFN-y/- ocorreu um aumento contínuo da carga bacilar com o transcurso da infecção, enquanto os camundongos IFN-y+/+ foram capazes de reduzir a carga bacilar no tecido hepático nos períodos mais tardios da análise. As alteraíões histopatológicas começam mais tardiamente nos camundongos IFN-y-/- (30 dias de pós-infecção). Estes formam granulomas sempre em menor número e tamanho que os IFN-y+/+. As alterações granulomatosas no fígado persistem até os 100 dias no camundongo IFN-y-l- e diminuem nos IFN-y+/+. Portanto, a presença do...

Instability and site-specific excision of integration-proficient mycobacteriophage L5 plasmids: development of stably maintained integrative vectors.

Integrative vectors expressing foreign genes are used as tools for the development of recombinant vaccines in mycobacteria since it is assumed that these vectors are stably maintained even without antibiotic selection. We here demonstrate that integration-proficient vectors are lost from the mycobacterial genome in high frequency. Loss of integrated vectors occurred in recA+ and in recA-strains, indicating a RecA-independent mechanism. Loss of the integrated vector was prevented when integrase gene function was carried on a separate plasmid that is unable to replicate in mycobacteria, indicating that excision is a function of integrase. By providing attP in cis and integrase function in trans, vectors integrating at the attB site are stably maintained, even when carrying genes that deleteriously affect the host.

[Application of rapid detection for Mycobacterium tuberculosis with phage splitting assay].

OBJECTIVE: To study the significance of rapid identification for Mycobacterium tuberculosis with phage splitting assay. METHODS: Strains of Mycobacterium tuberculosis, non-tuberculosis mycobacterium, non-mycobacterium and samples of sputum with pulmonary tuberculosis were rapidly detected by phage spot technique. RESULTS: The strains of Mycobacterium tuberculosis H37Rv, bovis and africanum were all positive. The results of 10 strains of non-tuberculosis mycobacterium and 7 strains of non-mycobacterium were negative. All of 30 clinical isolates from the patients of the pulmonary tuberculosis were positive. 19 of 20 sputum specimen of pulmonary tuberculosis, which were all positive detected by smear and culture, were positive. There were 15 specimen positive in 21 sputum with negative tested by smear and positive by culture. Besides, 5 of 19 sputum specimen with negative by smear and culture were positive detected by this method. CONCLUSION: The phage splitting assay can be used for rapid identification of Mycobacterium tuberculosis, which possesses high specificity and sensitivity for detection of Mycobacterium tuberculosis in sputum specimen.

Protein-DNA complexes in mycobacteriophage L5 integrative recombination.

The temperate mycobacteriophage L5 integrates site specifically into the genomes of Mycobacterium smegmatis, Mycobacterium tuberculosis, and Mycobacterium bovis bacillus Calmette-Guérin. This integrative recombination event occurs between the phage L5 attP site and the mycobacterial attB site and requires the phage-encoded integrase and mycobacterial-encoded integration host factor mIHF. Here we show that attP, Int-L5, and mIHF assemble into a recombinationally active complex, the intasome, which is capable of attB capture and formation of products. The arm-type integrase binding sites within attP play specialized roles in the formation of specific protein-DNA architectures; the intasome is constructed by the formation of intramolecular integrase bridges between one pair of sites, P4-P5, and the attP core, while an additional pair of sites, P1-P2, is required for interaction with attB.

Conditionally replicating luciferase reporter phages: improved sensitivity for rapid detection and assessment of drug susceptibility of Mycobacterium tuberculosis.

TM4 is a lytic mycobacteriophage which infects mycobacteria of clinical importance. A luciferase reporter phage, phAE40, has been constructed from TM4 and was previously shown to be useful for the rapid detection and drug susceptibility testing of Mycobacterium tuberculosis. However, the lytic nature of the phage results in a loss of detectable light output and limits the sensitivity of detection. We describe several strategies aimed at improving the luciferase activity generated by TM4 luciferase phages, including (i) varying the position of the luciferase gene in the phage genome, (ii) isolating host-range mutants of the phage, and (iii) introducing temperature-sensitive mutations in the phage such that it will not replicate at the infecting temperature. Several new phages generated by these methods show increased intensity of luciferase production compared to the first-generation reporter phage phAE40, and one phage, phAE88, also demonstrates an enhanced duration of luciferase activity. This has allowed the detection of as few as 120 BCG cells and the determination of drug susceptibilities of M. tuberculosis in as little as 1 day.

Mycobacteriophage L5 infection of Mycobacterium bovis BCG: implications for phage genetics in the slow-growing mycobacteria.

Mycobacteriophage L5 is a well-characterized temperate phage that forms stable lysogens in Mycobacterium smegmatis. The host range of L5 is, however, unclear because previous reports suggested that it does not infect slow-growing mycobacteria such as Mycobacterium tuberculosis and bacille Calmette-Guerin (BCG). Moreover, luciferase reporter phage derivatives of L5 failed to produce light from BCG, suggesting that infection is blocked at or before the stage of DNA injection. In this study, we demonstrate that L5 infection of slow growing mycobacteria specifically requires a high concentration of Ca2+, conditions that differs from those required for infection of M. smegmatis by L5 and for infection of BCG by the closely related phage D29. In addition, we show that there are specific genetic determinants of L5 that confer the ability to infect slow growing mycobacteria, without altering infection of M. smegmatis. These observations extend the use of phage L5 for the diagnosis and analysis of tuberculosis and other mycobacterial diseases.

Bacteriophages as tools for vaccine development.

The construction of live recombinant bacterial vaccines requires a reasonably sophisticated genetic system for the introduction, stabilization and expression of foreign antigen genes. Bacteriophages offer a rich collection of tools that can be used for vaccine construction, including site-specific integration-proficient vectors, non-antibiotic selectable markers and signals for efficient transcription and translation of foreign genes. We describe the characterization of a temperate phage of the mycobacteria, mycobacteriophage L5, and application of these phage studies for the construction of recombinant BCG vaccines.