Mycobacterium tuberculosis (MTB) is the principal cause of human tuberculosis (TB), which is a serious health problem worldwide. The development of innovative therapeutic modalities to treat TB is mainly due to the emergence of multi drug resistant (MDR) TB. Autophagy is a cell-host defense process. Previous studies have reported that autophagy-activating agents eliminate intracellular MDR MTB. Thus, combining a direct antibiotic activity against circulating bacteria with autophagy activation to eliminate bacteria residing inside cells could treat MDR TB. We show that the synthetic peptide, IP-1 (KFLNRFWHWLQLKPGQPMY), induced autophagy in HEK293T cells and macrophages at a low dose (10 μM), while increasing the dose (50 μM) induced cell death; IP-1 induced the secretion of TNFα in macrophages and killed Mtb at a dose where macrophages are not killed by IP-1. Moreover, IP-1 showed significant therapeutic activity in a mice model of progressive pulmonary TB. In terms of the mechanism of action, IP-1 sequesters ATP in vitro and inside living cells. Thus, IP-1 is the first antimicrobial peptide that eliminates MDR MTB infection by combining four activities: reducing ATP levels, bactericidal activity, autophagy activation, and TNFα secretion.

Background: Tuberculosis is the leading cause of death by an infectious microorganism worldwide. Conventional treatment lasts at least six months and has adverse effects; therefore, it is important to find therapeutic alternatives that reduce the bacterial load and may reduce the treatment duration. The immune response against tuberculosis can be modulated by several mechanisms, including extracellular vesicles (EVs), which are nano-sized membrane-bound structures that constitute an efficient communication mechanism among immune cells. Methods: The EVs released by the J774A.1 mouse macrophage cell line, both spontaneously (S-EV) and after infection with Mycobacterium tuberculosis H37Rv (Mtb-EV), were purified by ultra-centrifugation and size-exclusion chromatography. The size distribution and chemical composition of these EVs were evaluated, and their effect on the bacterial load and the production of cytokines was determined in both in vitro and in vivo models of M. tuberculosis infection. Results: Mtb-EV are larger than S-EV, they contain M. tuberculosis-specific antigens (not detected in EVs released from M. fortuitum-infected J774A.1 cells) and are rich in phosphatidylserine, present in their outer membrane layer. S-EV, but not Mtb-EV, reduced the bacterial load and the production of MCP-1 and TNF-α in M. tuberculosis-infected macrophages, and these effects were reversed when phosphatidylserine was blocked with annexin V. Both S-EV and Mtb-EV significantly reduced the lung bacterial load in mice infected with M. tuberculosis after 60 days of treatment, but they had no effect on survival or on the lung pneumonic area of these mice. Conclusion: J774A.1 macrophages infected with M. tuberculosis H37Rv released EVs that differed in size and phosphatidylserine content from spontaneously released EVs, and these EVs also had different biological effects: S-EV reduced the mycobacterial load and the cytokine production in vitro (through a phosphatidylserine-dependent mechanism), while both EVs reduced the lung bacterial load in vivo. These results are the basis for further experiments to evaluate whether EVs improve the efficiency of the conventional treatment for tuberculosis.

Tuberculosis (TB) is a chronic infectious disease in which prolonged, non-resolutive inflammation of the lung may lead to metabolic and neuroendocrine dysfunction. Previous studies have reported that individuals coursing pulmonary TB experience cognitive or behavioural changes; however, the pathogenic substrate of such manifestations have remained unknown. Here, using a mouse model of progressive pulmonary TB, we report that, even in the absence of brain infection, TB is associated with marked increased synthesis of both inflammatory and anti-inflammatory cytokines in discrete brain areas such as the hypothalamus, the hippocampal formation and cerebellum accompanied by substantial changes in the synthesis of neurotransmitters. Moreover, histopathological findings of neurodegeneration and neuronal death were found as infection progressed with activation of p38, JNK and reduction in the BDNF levels. Finally, we perform behavioural analysis in infected mice throughout the infection, and our data show that the cytokine and neurochemical changes were associated with a marked onset of cognitive impairment as well as depressive- and anxiety-like behaviour. Altogether, our results suggest that besides pulmonary damage, TB is accompanied by an extensive neuroinflammatory and neurodegenerative state which explains some of the behavioural abnormalities found in TB patients.

Tuberculosis (TB) is one of the ten leading causes of death worldwide. Patients with TB have been observed to suffer from depression and anxiety linked to social variables. Previous experiments found that the substantial pulmonary inflammation associated with TB causes neuroinflammation, neuronal death, and behavioral impairments in the absence of brain infection. Curcumin (CUR) is a natural product with antioxidant, anti-inflammatory and antibacterial activities. In this work, we evaluated the CUR effect on the growth control of mycobacteria in the lungs and the anti-inflammatory effect in the brain using a model of progressive pulmonary TB in BALB/c mice infected with drug-sensitive mycobacteria (strain H37Rv). The results have shown that CUR decreased lung bacilli load and pneumonia of infected animals. Finally, CUR significantly decreased neuroinflammation (expression of TNF$\mathsf{\alpha }$, IFN$\mathsf{\gamma }$ and IL12) and slightly increased the levels of nuclear factor erythroid 2-related to factor 2 (Nrf2) and the brain-derived neurotrophic factor (BDNF) levels, improving behavioral status. These results suggest that CUR has a bactericidal effect and can control pulmonary mycobacterial infection and reduce neuroinflammation. It seems that CUR has a promising potential as adjuvant therapy in TB treatment.

The study of host-pathogen interactions using in vivo models with intracellular pathogens like Mycobacterium tuberculosis (Mtb) entails technical limitations, such as: (i) Selecting an efficient differential lysis system to enrich the pathogen cells; (ii) obtaining sufficient high-quality RNA; and (iii) achieving an efficient rRNA depletion. Thus, some authors had used flow cytometers to separate infected cells or significantly increase the sequencing depth of host–pathogen RNA libraries to observe the pathogens’ gene expression. However, these options carry additional expenses in specialized equipment typically not available for all laboratories. Here, we propose an experimental protocol involving differential cell lysis and a probe-based ribosomal depletion to determine the gene expression of Mtb and its host during in vivo infection. This method increased the number of observed pathogen-expressed genes from 13 using the traditional RNA-seq approach to 702. After eliminating rRNA reads, we observed that 61.59% of Mtb sequences represented 702 genes, while 38.41% represented intergenic regions. Some of the most expressed genes codified for IS1081 (Rv2512c) transposase and eight PE-PGRS members, such as PGRS49 and PGRS50. As expected, a critical percent of the expressed genes codified for secreted proteins essential for infection, such as PE68, lppN, and LpqH. Moreover, three Mtb ncRNAs were highly expressed (small RNA MTS2823, transfer-messenger RNA RF00023, and ribozyme RF00010). Many of the host-expressed genes were related to the inflammation process and the expression of surfactant proteins such as the Sftpa and Sftpc, known to bind Mtb to alveolar macrophages and mi638, a microRNA with no previous associations with pulmonary diseases. The main objective of this study is to present the method, and a general catalog of the Mtb expressed genes at one point of the in vivo infection. We believe our method represents a different approach to the existing ones to study host–pathogen interactions in tuberculosis and other similar intracellular infections, without the necessity of specialized equipment.