Selective host autophagy is induced during the intracellular parasite Toxoplasma gondii infection controlling amino acid levels.

Toxoplasma gondii, a widespread parasite, has the ability to infect nearly any nucleated cell in warm-blooded vertebrates. It is estimated that around 2 billion people globally have been infected by this pathogen. Although most healthy individuals can effectively control parasite replication, certain parasites may evade the immune response, establishing cysts in the brain that are refractory to the immune system and resistant to available drugs. For its chronic persistence in the brain, the parasite relies on host cells' nutrients, particularly amino acids and lipids. Therefore, understanding how latent parasites persist in the brain is crucial for identifying potential drug targets against chronic forms. While shielded within parasitophorous vacuoles (PVs) or cysts, Toxoplasma exploits the host endoplasmic reticulum (ER) metabolism to sustain its persistence in the brain, resulting in host neurological alterations. In this study, we demonstrate that T. gondii disrupts the host ER homeostasis, resulting in the accumulation of unfolded protein within the host ER. The host counters this stress by initiating an autophagic pathway known as ER-phagy, which breaks down unfolded proteins into amino acids, promoting their recycling. Our findings unveil the underlying mechanisms employed by T. gondii to exploit host ER and lysosomal pathways, enhancing nutrient levels during infection. These insights provide new strategies for the treatment of toxoplasmosis. IMPORTANCE: Intracellular parasites employ several mechanisms to manipulate the cellular environment, enabling them to persist in the host. Toxoplasma gondii, a single-celled parasite, possesses the ability to infect virtually any nucleated cell of warm-blooded vertebrates, including nearly 2 billion people worldwide. Unfortunately, existing treatments and immune responses are not entirely effective in eliminating the chronic persisting forms of the parasite. This study reveals that T. gondii induces the host's autophagic pathway to boost amino acid levels in infected cells. The depletion of amino acids, in turn, influences the persistence of the parasite's chronic forms. Significantly, our investigation establishes the crucial role of host endoplasmic reticulum (ER)-phagy in the parasite's persistence within the host during latent infection.

Host autophagy is exploited by the intracellular parasite Toxoplasma gondii to enhance amino acids levels.

Toxoplasma gondii, a widespread parasite, has the ability to infect nearly any nucleated cell in warm-blooded vertebrates. It is estimated that around 2 billion people globally have been infected by this pathogen. Although most healthy individuals can effectively control parasite replication, certain parasites may evade the immune response, establishing cysts in the brain that are refractory to the immune system and resistance to available drugs. For its chronic persistence in the brain, the parasite relies on host cells' nutrients, particularly amino acids and lipids. Therefore, understanding how latent parasites persist in the brain is crucial for identifying potential drug targets against chronic forms. While shielded within parasitophorous vacuoles (PVs) or cysts, Toxoplasma exploits the host endoplasmic reticulum (ER) metabolism to sustains its persistence in the brain, resulting in host neurological alterations. In this study, we demonstrate that T. gondii disrupts the host ER homeostasis, resulting in accumulation of unfolded protein with the host ER. The host counters this stress by initiating an autophagic pathway known as ER-phagy, which breaks down unfolded proteins into amino acids, promoting their recycling. Remarkably, the persistence of latent forms in cell culture as well as behavioral changes in mice caused by the latent infection could be successfully reversed by restricting the availability of various amino acids during T. gondi infection. Our findings unveil the underlying mechanisms employed by T. gondii to exploit host ER and lysosomal pathways, enhancing nutrient levels during infection. These insights provide new strategies for the treatment of toxoplasmosis. Importance: Intracellular parasites employ several mechanisms to manipulate the cellular environment, enabling them to persist in the host. Toxoplasma gondii , a single-celled parasite, possesses the ability to infect virtually any nucleated cell of warm-blooded vertebrates, including nearly 2 billion people worldwide. Unfortunately, existing treatments and immune responses are not entirely effective in eliminating the chronic persisting forms of the parasite. This study reveals that T. gondii induces the host's autophagic pathway to boost amino acid levels in infected cells. The depletion of amino acids, in turn, influences the persistence of the parasite's chronic forms, resulting in a reduction of neurological alterations caused by chronic infection in mice. Significantly, our investigation establishes the crucial role of host ER-phagy in the parasite's persistence within the host during latent infection.

Establishing the intracellular niche of obligate intracellular vacuolar pathogens.

Obligate intracellular pathogens occupy one of two niches - free in the host cell cytoplasm or confined in a membrane-bound vacuole. Pathogens occupying membrane-bound vacuoles are sequestered from the innate immune system and have an extra layer of protection from antimicrobial drugs. However, this lifestyle presents several challenges. First, the bacteria must obtain membrane or membrane components to support vacuole expansion and provide space for the increasing bacteria numbers during the log phase of replication. Second, the vacuole microenvironment must be suitable for the unique metabolic needs of the pathogen. Third, as most obligate intracellular bacterial pathogens have undergone genomic reduction and are not capable of full metabolic independence, the bacteria must have mechanisms to obtain essential nutrients and resources from the host cell. Finally, because they are separated from the host cell by the vacuole membrane, the bacteria must possess mechanisms to manipulate the host cell, typically through a specialized secretion system which crosses the vacuole membrane. While there are common themes, each bacterial pathogen utilizes unique approach to establishing and maintaining their intracellular niches. In this review, we focus on the vacuole-bound intracellular niches of Anaplasma phagocytophilum, Ehrlichia chaffeensis, Chlamydia trachomatis, and Coxiella burnetii.

Coxiella burnetii actively blocks IL-17-induced oxidative stress in macrophages.

Coxiella burnetii is a highly infectious pathogen that causes Q fever, a leading cause of culture-negative endocarditis. Coxiella first targets alveolar macrophages and forms a phagolysosome-like compartment called the Coxiella-Containing Vacuole (CCV). Successful host cell infection requires the Type 4B Secretion System (T4BSS), which translocates bacterial effector proteins across the CCV membrane into the host cytoplasm, where they manipulate numerous cell processes. Our prior transcriptional studies revealed that Coxiella T4BSS blocks IL-17 signaling in macrophages. Given that IL-17 is known to protect against pulmonary pathogens, we hypothesize that C. burnetii T4BSS downregulates intracellular IL-17 signaling to evade the host immune response and promote bacterial pathogenesis. Using a stable IL-17 promoter reporter cell line, we confirmed that Coxiella T4BSS blocks IL-17 transcription activation. Assessment of the phosphorylation state of NF-κB, MAPK, and JNK revealed that Coxiella downregulates IL-17 activation of these proteins. Using ACT1 knockdown and IL-17RA or TRAF6 knockout cells, we next determined that IL17RA-ACT1-TRAF6 pathway is essential for the IL-17 bactericidal effect in macrophages. In addition, macrophages stimulated with IL-17 generate higher levels of reactive oxygen species, which is likely connected to the bactericidal effect of IL-17. However, C. burnetii T4SS effector proteins block the IL-17-mediated oxidative stress, suggesting that Coxiella blocks IL-17 signaling to avoid direct killing by the macrophages.

The novel bacterial effector protein CbEPF1 mediates ER-LD membrane contacts to regulate host lipid droplet metabolism.

Effective intracellular communication between cellular organelles is pivotal for maintaining cellular homeostasis. Tether proteins, which are responsible for establishing membrane contact sites between cell organelles, enable direct communication between organelles and ultimately influence organelle function and host cell homeostasis. While recent research has identified tether proteins in several bacterial pathogens, their functions have predominantly been associated with mediating inter-organelle communication specifically between the bacteria containing vacuole (BCV) and the host endoplasmic reticulum (ER). However, this study reveals a novel bacterial effector protein, CbEPF1, which acts as a molecular tether beyond the confines of the BCV and facilitates interactions between host cell organelles. Coxiella burnetii, an obligate intracellular bacterial pathogen, encodes the FFAT motif-containing protein CbEPF1 which localizes to host lipid droplets (LDs). CbEPF1 establishes inter-organelle contact sites between host LDs and the ER through its interactions with VAP family proteins. Intriguingly, CbEPF1 modulates growth of host LDs in a FFAT motif-dependent manner. These findings highlight the potential for bacterial effector proteins to impact host cellular homeostasis by manipulating inter-organelle communication beyond conventional BCVs.

Ectopic Expression of Rv0023 Mediates Isoniazid/Ethionamide Tolerance via Altering NADH/NAD+ Levels in Mycobacterium smegmatis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) accounts for nearly 1.2 million deaths per annum worldwide. Due to the emergence of multidrug-resistant (MDR) Mtb strains, TB, a curable and avertable disease, remains one of the leading causes of morbidity and mortality. Isoniazid (INH) is a first-line anti-TB drug while ethionamide (ETH) is used as a second-line anti-TB drug. INH and ETH resistance develop through a network of genes involved in various biosynthetic pathways. In this study, we identified Rv0023, an Mtb protein belonging to the xenobiotic response element (XRE) family of transcription regulators, which has a role in generating higher tolerance toward INH and ETH in Mycobacterium smegmatis (Msmeg). Overexpression of Rv0023 in Msmeg leads to the development of INH- and ETH-tolerant strains. The strains expressing Rv0023 have a higher ratio of NADH/NAD+, and this physiological event is known to play a crucial role in the development of INH/ETH co-resistance in Msmeg. Gene expression analysis of some target genes revealed reduction in the expression of the ndh gene, but no direct interaction was observed between Rv0023 and the ndh promoter region. Rv0023 is divergently expressed to Rv0022c (whiB5) and we observed a direct interaction between the recombinant Rv0023 protein with the upstream region of Rv0022c, confirmed using reporter constructs of Msmeg. However, we found no indication that this interaction might play a role in the development of INH/ETH drug tolerance.

Mce2R/Rv0586 of Mycobacterium tuberculosis is the functional homologue of FadRE. coli.

Lipid metabolism is critical to Mycobacterium tuberculosis survival and infection. Unlike Escherichia coli, which has a single FadR, the M. tuberculosis genome encodes five proteins of the FadR sub-family. While the role of E. coli FadR as a regulator of fatty acid metabolism is well known, the definitive functions of M. tuberculosis FadR proteins are still under investigation. An interesting question about the M. tuberculosis FadRs remains open: which one of these proteins is the functional homologue of E. coli FadR? To address this, we have applied two different approaches. The first one was the bioinformatics approach and the second one was the classical molecular genetic approach involving complementation studies. Surprisingly, the results of these two approaches did not agree. Among the five M. tuberculosis FadRs, Rv0494 shared the highest sequence similarity with FadRE. coli and Rv0586 was the second best match. However, only Rv0586, but not Rv0494, could complement E. coli ∆fadR, indicating that Rv0586 is the M. tuberculosis functional homologue of FadRE. coli. Further studies showed that both regulators, Rv0494 and Rv0586, show similar responsiveness to LCFA, and have conserved critical residues for DNA binding. However, analysis of the operator site indicated that the inter-palindromic distance required for DNA binding differs for the two regulators. The differences in the binding site selection helped in the success of Rv0586 binding to fadB upstream over Rv0494 and may have played a critical role in complementing E. coli ∆fadR. Further, for the first time, we report the lipid-responsive nature of Rv0586.

An IclR like protein from mycobacteria regulates leuCD operon and induces dormancy-like growth arrest in Mycobacterium smegmatis.

leuCD operon encodes isopropylmalate isomerase (IPMI), an essential enzyme in leucine biosynthesis. Leucine biosynthesis is one of the essential metabolic pathways for Mycobacterium tuberculosis survival inside the macrophage. In this study, we identified an IclR like transcription regulator, Rv2989 involved in regulation of leuCD expression. Further, we have shown that the Rv2989 binding site overlaps with the promoter region of leuCD, indicating its direct involvement in the regulation of this operon. Ectopic expression of Rv2989 in M. smegmatis induced growth arrest with significantly decreased levels of leuCD transcript. However, supplementation with leucine could not reverse the growth arrest, suggesting the involvement of Rv2989 in the regulation of other essential pathways. Growth-arrested cells were elongated, had lost acid fastness and accumulated lipid droplets similar to a dormancy-like state. In conclusion, the Rv2989 expression has pleiotropic effects on M. smegmatis. It negatively regulates leuCD operon and induces dormancy-like growth arrest.

Rv0494 is a starvation-inducible, auto-regulatory FadR-like regulator from Mycobacterium tuberculosis.

Fatty acid metabolism plays an important role in the survival and pathogenesis of Mycobacterium tuberculosis. Lipids are assumed to be the major source of energy during dormancy. Here, we report the characterization of a starvation-inducible, lipid-responsive transcriptional regulator, Rv0494, divergently transcribed from the Rv0493c probable operon. The striking difference in the transcriptional regulatory apparatus between mycobacteria and other well-studied organisms, such as Escherichia coli, is the organization of mycobacterial promoters. Mycobacterial promoters have diverse architectures and most of these promoters function inefficiently in E. coli. In this study, we characterized the promoter elements of Rv0494 along with the sigma factors required for transcription initiation. Rv0494 promoter activity increased under nutrient starvation conditions and was transcribed via two promoters: the promoter proximal to the translational start site was active under standard growth conditions, whilst both promoters contributed to the increased activity seen during starvation, with the major contribution from the distal promoter. Furthermore, Rv0494 translation initiated at a codon located 9 bp downstream of the annotated start codon. Rv0494 bound to its upstream sequence to auto-regulate its own expression; this binding was responsive to long-chain fatty acyl-CoA molecules. We further report Rv0494-mediated transcriptional regulation of the Rv2326c gene - a probable transmembrane ATP-binding transporter encoding gene.

Role of cellular senescence in hepatic wound healing and carcinogenesis.

A state of permanent growth arrest characterises a senescent cell. Both the beneficial and deleterious effects that have accrued in senescent cells are observed in a complex organ, such as the liver. Injury to liver tissues triggers processes of regeneration and associated wound healing. Persistent injury can also lead to the neoplastic state. Recent evidence linked the senescent characteristics of the cells to the beneficial processes of wound healing and tumour surveillance in the liver. On the other hand, the secretory phenotype of senescent cells can also selectively promote undesirable neoplastic progression. In an evolutionary context, a senescent cell can function primarily as an adaptive response featuring the characteristics of altruism, trade-offs and bystander effects. Using the liver cell as a model system, this review focuses on the current knowledge of the role of senescence in these seemingly contradictory cell phenomena.