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Adaptation of selected mycobacteria during re-infection.A. Diagram showing the strategy followed in the re-infection experiments.B. Equal amounts of ancestral (ANC) and selected (SEL) bacteria were used to re-infect RAW 264.7 macrophages as indicated in experimental procedures. After 1, 2 and 3 days of infection, the number of cfu were calculated.C. Ancestral and selected bacteria were used to re-infect primary bone marrow macrophages (BMMs) as indicated in Experimental procedures. After 1, 2, 3 and 7 days of infection, the number of colonies was counted and cfu were calculated.Data represent the mean ± S.E.M., (**) P ≤ 0.01 from two-tailed Student's t-test from three independent experiments.D. Quantitative PCR (qPCR) analysis of the expression of selected M. bovis BCG genes. Data represent the mean ± SD from four independent experiments. Student's t-tests were performed to verify the differences in the fold change in gene expression.E. Lung bacillary loads in BALB/c mice after intratracheal inoculation (1.25 × 105 cfu) of selected (red bars) or ancestral bacteria (green bars). The data represent the mean number of cfu ± SD in five mice of one representative experiment out of two independent experiments, (**) P ≤ 0.01 from two-tailed Student's t-test.F. Survival of Nude mice after intratracheal inoculation with (1.25 × 104 cfu) of selected (red bars) or ancestral (green bars) bacteria. Statistical analysis for survival curves was performed using Mantel-Cox test P ≤ 0.0005.
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Some intracellular bacteria are known to cause long-term infections that last decades without compromising the viability of the host. Although of critical importance, the adaptations that intracellular bacteria undergo during this long process of residence in a host cell environment remain obscure. Here, we report a novel experimental approach to s...
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Mycobacterium bovis is an intracellular pathogen that causes tuberculosis in cattle. Following infection, the pathogen resides and persists inside host macrophages by subverting host immune responses via a diverse range of mechanisms. Here, a high-density bovine microarray platform was used to examine the bovine monocyte-derived macrophage transcri...
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... The typical Mtb life cycle includes a phase of the primary disease followed by a stage of longterm occult and persistent intracellular infection. During this "latent" period, bacilli survive in a non-vegetative dormant form characterized by a thick lipid-rich bacterial cell wall and lipid accumulations (Vaźquez et al., 2014) protecting from the degrading activity of host autophagolysosomal enzymes (Figure 1). Alveolar macrophages serve as the first port of entry and primary host for Mtb, killing it and presenting antigens while also sheltering persistent bacilli (van Crevel et al., 2003). ...
... The immu ne resp onse to mycoba cterial e arly-conserved immunodominant epitopes of the Esx secretion system triggers pH to drop to 5.0 in the phagolysosome. However, the time gained allows Mtb to alter its ability to use carbon sources with the preference of glucose usage and storage of neutral lipids (triacylglycerols (TAG)) in droplets (Vaźquez et al., 2014) followed by mycobacterial entry into the dormant phase. ...
MHC class I antigen processing is an underappreciated area of nonviral host–pathogen interactions, bridging both immunology and cell biology, where the pathogen’s natural life cycle involves little presence in the cytoplasm. The effective response to MHC-I foreign antigen presentation is not only cell death but also phenotypic changes in other cells and stimulation of the memory cells ready for the next antigen reoccurrence. This review looks at the MHC-I antigen processing pathway and potential alternative sources of the antigens, focusing on Mycobacterium tuberculosis (Mtb) as an intracellular pathogen that co-evolved with humans and developed an array of decoy strategies to survive in a hostile environment by manipulating host immunity to its own advantage. As that happens via the selective antigen presentation process, reinforcement of the effective antigen recognition on MHC-I molecules may stimulate subsets of effector cells that act earlier and more locally. Vaccines against tuberculosis (TB) could potentially eliminate this disease, yet their development has been slow, and success is limited in the context of this global disease’s spread. This review’s conclusions set out potential directions for MHC-I-focused approaches for the next generation of vaccines.
... The growth characteristics differed for the isolates with BCG-lung displaying diminished growth in culture media compared to BCG-vaccine and BCG-brain. Fitness loss for growth in vitro was shown to correlate with adaptive survival inside the macrophages [39]. Work in M. tuberculosis has uncovered mechanisms to permit the progression of disease, such as changes in lipid metabolism and transcription regulation [4]. ...
We characterized Mycobacterium bovis BCG isolates found in lung and brain samples from a previously vaccinated patient with IFNγR1 deficiency. The isolates collected displayed distinct genomic and phenotypic features consistent with host adaptation and associated changes in antibiotic susceptibility and virulence traits.
Background: We report a case of a patient with partial recessive IFNγR1 deficiency who developed disseminated BCG infection after neonatal vaccination (BCG-vaccine). Distinct M. bovis BCG-vaccine derived clinical strains were recovered from the patient’s lungs and brain.
Methods: BCG strains were phenotypically (growth, antibiotic susceptibility, lipid) and genetically (whole genome sequencing) characterized. Mycobacteria cell infection models were used to assess apoptosis, necrosis, cytokine release, autophagy, and JAK-STAT signaling.
Results: Clinical isolates BCG-brain and BCG-lung showed distinct Rv0667 rpoB mutations conferring high- and low-level rifampin resistance; the latter displayed clofazimine resistance through Rv0678 gene (MarR-like transcriptional regulator) mutations. BCG-brain and BCG-lung showed mutations in fadA2, fadE5, and mymA operon genes, respectively. Lipid profiles revealed reduced levels of PDIM in BCG-brain and BCG-lung and increased TAGs and Mycolic acid components in BCG-lung, compared to parent BCG-vaccine. In vitro infected cells showed that the BCG-lung induced a higher cytokine release, necrosis, and cell-associated bacterial load effect when compared to BCG-brain; conversely, both strains inhibited apoptosis and altered JAK-STAT signaling.
Conclusions: During a chronic-disseminated BCG infection, BCG strains can evolve independently at different sites likely due to particular microenvironment features leading to differential antibiotic resistance, virulence traits resulting in dissimilar responses in different host tissues.
... In the context of infections and at the commensal-pathogen transition, a major selective pressure is the innate immune system, with macrophages (MΦs) being key in the response to microbial infections (Wynn, Chawla, & Pollard, 2013). Due to this role of MΦs, several studies have recently used experimental adaptation to understand how yeast or bacteria evolve under MΦ attack (Azevedo et al., 2016;Ensminger, Yassin, Miron, & Isberg, 2012;Vázquez et al., 2014;Wartenberg et al., 2014). Recently, we used this approach to study the adaptation of a commensal E. coli to the interaction with MΦs . ...
Small-colony variants (SCVs) are commonly observed in evolution experiments and clinical isolates, being associated with antibiotic resistance and persistent infections. We recently observed the repeated emergence of Escherichia coli SCVs during adaptation to the interaction with macrophages. To identify the genetic targets underlying the emergence of this clinically relevant morphotype, we performed whole-genome sequencing of independently evolved SCV clones. We uncovered novel mutational targets, not previously associated with SCVs (e.g. cydA, pepP) and observed widespread functional parallelism. All SCV clones had mutations in genes related to the electron-transport chain. As SCVs emerged during adaptation to macrophages, and often show increased antibiotic resistance, we measured SCV fitness inside macrophages and measured their antibiotic resistance profiles. SCVs had a fitness advantage inside macrophages and showed increased aminoglycoside resistance in vitro, but had collateral sensitivity to other antibiotics (e.g. tetracycline). Importantly, we observed similar results in vivo. SCVs had a fitness advantage upon colonization of the mouse gut, which could be tuned by antibiotic treatment: kanamycin (aminoglycoside) increased SCV fitness, but tetracycline strongly reduced it. Our results highlight the power of using experimental evolution as the basis for identifying the causes and consequences of adaptation during host-microbe interactions.
... Densitometry analysis was performed by ImageJ to quantify PPM1A band intensities as normalized to α-tubulin or GAPDH, and fold changes relative to day 0 post infection are reported. aggregate infection model would reproduce reports that Mtb can viably persist in macrophages ( Figure 1E, 1F) [49,50]. ...
Co-infection with HIV-1 and Mycobacterium tuberculosis (Mtb) is a major public health issue. While some research has described how each pathogen accelerates the course of infection of the other pathogen by compromising the immune system, very little is known about the molecular biology of HIV-1/Mtb co-infection at the host cell level. This is somewhat surprising, as both pathogens are known to replicate and persist in macrophages. We here identify Protein Phosphatase, Mg2+/Mn2+-dependent 1A (PPM1A) as a molecular link between Mtb infection and increased HIV-1 susceptibility of macrophages. We demonstrate that both Mtb and HIV-1 infection induce the expression of PPM1A in primary human monocyte/macrophages and THP-1 cells. Genetic manipulation studies revealed that increased PPMA1 expression rendered THP-1 cells highly susceptible to HIV-1 infection, while depletion of PPM1A rendered them relatively resistant to HIV-1 infection. At the same time, increased PPM1A expression abrogated the ability of THP-1 cells to respond to relevant bacterial stimuli with a proper cytokine/chemokine secretion response, blocked their chemotactic response and impaired their ability to phagocytose bacteria. These data suggest that PPM1A, which had previously been shown to play a role in the antiviral response to Herpes Simplex virus infection, also governs the antibacterial response of macrophages to bacteria, or at least to Mtb infection. PPM1A thus seems to play a central role in the innate immune response of macrophages, implying that host directed therapies targeting PPM1A could be highly beneficial, in particular for HIV/Mtb co-infected patients.
... However, our results provide an explanation for the observation that clinical isolates of M. tuberculosis manipulate autophagy for their survival (40) and mirror those seen in M. tuberculosis infection of human primary dendritic cells (41), human type II alveolar epithelial cells (42), and some primary alveolar macrophages from adult macaques (43). Consistent with our observations, in Listeria-infected macrophages, bacteria are localized to both the cytosol and LC3 + compartments in which bacteria grow (44), and long-term infection of macrophages with M. bovis BCG also leads to growth of the bacteria in LC3 + compartments (45). ...
In extrapulmonary tuberculosis, the most common site of infection is within the lymphatic system, and there is growing recognition that lymphatic endothelial cells (LECs) are involved in immune function. Here, we identified LECs, which line the lymphatic vessels, as a niche for Mycobacterium tuberculosis in the lymph nodes of patients with tuberculosis. In cultured primary human LECs (hLECs), we determined that M. tuberculosis replicates both in the cytosol and within autophagosomes, but the bacteria failed to replicate when the virulence locus RD1 was deleted. Activation by IFN-γ induced a cell-autonomous response in hLECs via autophagy and NO production that restricted M. tuberculosis growth. Thus, depending on the activation status of LECs, autophagy can both promote and restrict replication. Together, these findings reveal a previously unrecognized role for hLECs and autophagy in tuberculosis pathogenesis and suggest that hLECs are a potential niche for M. tuberculosis that allows establishment of persistent infection in lymph nodes.
