The serine/threonine protein kinase F (PknF) of Mycobacterium tuberculosis (Mtb) has poorly defined targets and functions but is involved in limiting NLRP3 inflammasome activation in murine macrophages and dendritic cells in vitro . The importance of PknF for the virulence of Mtb in vivo is not known. Here, we demonstrate that the Mtb CDC1551 deletion mutant of pknF ( Δ pknF ) expresses significantly increased levels of the lipid pthiocerol dimycoserosate (PDIM), a polyketide lipid with pro-virulence properties. The Δ pknF mutant strain, when compared to the Mtb and complemented strains, had a 100-fold increase in growth at day 28 and about a 10-fold increase in growth at days 90-98 in the lungs of mice. The increase in pulmonary bacterial loads after infection with Δ pknF strain was conserved even in Nlrp3 -deficient mice, arguing that PknF modulates Mtb virulence independently of NLRP3-inflammasome activation in mice. Staining of lung sections revealed increased inflammation in the lungs of Δ pknF strain-infected mice when compared to Mtb and the complemented mutant strain. Highly susceptible B6.Sst1 S mice displayed decreased host resistance with significantly decreased survival when infected with the Δ pknF strain compared to the complemented strain or Mtb. In conclusion, our data suggest that expression of PknF, as a modulator of multiple downstream effector proteins, restricts the virulence of Mtb in the lungs of mice through an NLRP3 inflammasome-independent mechanism but potentially via suppressing expression of the virulence lipid, PDIM. Author summary:The human pathogen Mycobacterium tuberculosis (Mtb) encodes 11 serine/threonine protein kinases (Pkn A-I,K and L). In vitro , PknF inhibits activation of innate immune responses in macrophages and dendritic cells. The importance of that protein for the virulence of the bacteria in the context of a live animal infection is unknown. We used a hypersusceptible mouse strain to analyze the impact of deleting the pknF gene on the virulence of the bacteria by monitoring the survival of the mice. We show that the Δ pknF deletion mutant kills mice faster than wild-type bacteria or Δ pknF mutant bacteria expressing a wild-type copy of the pknF gene (Δ pknF -C). Next, we analyzed the growth of the different bacterial strains in the infected mice and demonstrated that at day 28 and day 90-98 timepoints the Δ pknF mutant bacterial strains showed a significant increase in bacterial burden in the lungs, bronchoalveolar lavage fluid and the spleen. An analysis of the total lipids of the bacterial stocks showed that the Δ pknF Mtb bacteria increased expression of the virulence lipid, pthiocerol dimycoserosate (PDIM), suggesting a negative regulation of this lipid by PknF likely affecting bacterial virulence in the lungs of mice.

Ribosomes are central to protein synthesis and a frequent target for antibiotics. In fast-growing bacteria, the ribosome content is proportional to the growth rate; how ribosomes and protein synthesis are regulated during nutrient starvation remains poorly understood, particularly in single cells. To address this, we fluorescently labeled ribosomal proteins (RPs) in Salmonella and explored their variations and regulation in single cells. We show that the RP levels become heterogeneous during the transition to the stationary phase. Unexpectedly, cells with higher RP levels responded less to the induction of gene expression but accumulated more virulence gene products. Our work further reveals that adenosine 3 ',5 '-monophosphate (cAMP) signaling increases the heterogeneity of the levels of RPs and other gene products. Fluorescence dilution assay and proteomic analysis indicate that cAMP signaling promotes gene expression heterogeneity by directing proteome-wide adaptation, which enables growth heterogeneity, hence differential dilution of gene products during nutrient depletion.

Mycobacterium tuberculosis (Mtb) primarily infects human lung macrophages, which serve as its major replication niche. Mtb can manipulate host macrophage cell death pathways to its advantage by inhibiting apoptosis and inducing necrotic cell death. However, the specific necrotic cell death pathway activated in human macrophages after Mtb infection remains unclear. Here, we used the THP-1 cell line and primary human monocyte-derived macrophage (hMDM) to analyze multiple programmed cell death pathways during days 1-3 after Mtb infection. Confocal microscopic analysis demonstrates that Mtb-infected THP-1 cells or hMDMs rarely exhibited apoptosis. Immunoblotting shows that Mtb induces significant CASP3 and GSDME activation in THP-1 cells, but not in hMDMs. We show that Mtb, in THP-1 cells but not hMDM, induces a significant increase in GSDMD cleavage, a hallmark of pyroptosis. MLKL phosphorylation was not observed in THP-1 cells or hMDMs during Mtb infections, indicating an absence of necroptosis. No changes in ferroptosis markers such as GPX4 expression or lipid peroxidation levels were detected. Time-lapse live-cell imaging revealed no lysosomal membrane permeabilization prior to plasma membrane rupture (PMR). However, we observed DNA release from Mtb-infected THP-1 cells and hMDMs after PMR. The DNA released from THP-1 cells exhibits low levels of myeloperoxidase and histone H3 citrullination. High-resolution confocal imaging shows that Mtb is associated with the released DNA. We demonstrate that pyroptosis induction in THP-1 cells is dispensable for the DNA release and cell death induction. In conclusion, our results reveal that Mtb-triggered cell death in hMDMs bypasses canonical cell death pathways like apoptosis, pyroptosis, necroptosis, and ferroptosis. Instead, cell death in both THP-1 cells and hMDMs correlates with DNA release, potentially similar to NETosis in neutrophils.

ABSTRACT Mycobacterium tuberculosis (Mtb) has been known to evade host innate immunity by manipulating macrophage function. Interleukin-1β (IL-1β) is a cytokine secreted by macrophages as a consequence of inflammasome activation. Mtb can inhibit activation of the NLRP3 and AIM2 inflammasomes and subsequent pyroptosis. The capacity of Mtb to manipulate other types of inflammasomes is unknown. In this study, we investigated whether Mtb or the nontuberculous mycobacteria Mycobacterium kansasii (Mkan) and Mycobacterium smegmatis (Msmeg) can inhibit the NLRP1 or pyrin inflammasomes. We show that none of the mycobacteria consistently inhibit the NLRP1 inflammasome after it is activated by priming with lipopolysaccharide (LPS) and treatment with 1G244 in human macrophages. Similarly, neither Mtb nor the NTMs inhibit the pyrin inflammasome after it is activated by priming with LPS and treating with TcdB toxin from Clostridium difficile in human macrophages. In murine macrophages, Mkan shows inhibition only at 3 h post-infection, and Msmeg, only at 24 h post-infection, whereas no inhibition was observed at any time point for Mtb. In conclusion, our main findings are that Mtb is unable to inhibit the NLRP1 and the pyrin inflammasomes in human macrophages. IMPORTANCE The research focuses on the interaction of mycobacteria with BMDMs and hMDMs regarding NLRP1 and pyrin inflammasome inhibition. We think that these results point to insights into the pathways of pathogen recognition in macrophages, which may have broader implications for host defense. These results are technically sound and provide new information to the field by showing that various mycobacteria do not inhibit the NLRP1 and Pyrin inflammasomes, at least under our specific experimental settings. Important limitations to the study are its narrow scope and limited mechanistic depth.

Mycobacterium tuberculosis (Mtb) induces necrotic cell death of infected macrophages, which contributes to tissue necrosis and the progressive loss of lung function during tuberculosis. Mtb can induce multiple forms of programmed necrosis, including necroptosis, pyroptosis and ferroptosis. Concurrently, Mtb also inhibits apoptosis to prevent a host-beneficial cell death response. This paper will first provide an overview of the programmed cell death pathways relevant to Mtb infection. It will then discuss how Mtb activates and manipulates these pathways under different conditions, including a comparison of the findings across mouse, human, and zebrafish-derived macrophages.

ABSTRACT The NLRP11 protein is only expressed in primates and participates in the activation of the canonical NLRP3 and non-canonical NLRP3 inflammasome activation after infection with gram-negative bacteria. Here, we generated a series of defined NLRP11 deletion mutants to further analyze the role of NLRP11 in NLRP3 inflammasome activation. Like the complete NLRP11 deletion mutant (NLRP11−/−), the NLRP11 mutant lacking the NAIP, C2TA, HET-E, and TP1 (NACHT) and leucine-rich repeat (LRR) domains (NLRP11∆N_LRR) showed reduced activation of the canonical NLRP3 inflammasome, whereas a pyrin domain mutant (NLRP11∆PYD) had no effect on NLRP3 activation. The NLRP11−/− and NLRP11∆N_LRR mutants, but not the NLRP11∆PYD mutant, also displayed reduced activation of caspase-4 during infection with the intracytosolic, gram-negative pathogen Shigella flexneri. We found that the human-adapted, acid-fast pathogen Mycobacterium tuberculosis and the opportunistic pathogen Mycobacterium kansasii both activate the non-canonical NLRP11 inflammasome in a caspase-4/caspase-5-dependent pathway. In conclusion, we show that NLRP11 functions in the non-canonical caspase-4/caspase-5 inflammasome activation pathway and the canonical NLRP3 inflammasome pathway and that NLRP11 is required for full recognition of mycobacteria by each of these pathways. Our work extends the spectrum of bacterial pathogen recognition by the non-canonical NLRP11-caspase4/caspase-5 pathway beyond gram-negative bacteria.IMPORTANCEThe activation of inflammasome complexes plays a crucial role in intracellular pathogen detection. NLRP11 and caspase-4 are essential for recognizing lipopolysaccharide (LPS), a molecule found in gram-negative bacteria such as the human pathogens Shigella spp., which activate both canonical NLRP3 and non-canonical inflammasome pathways. Through a series of deletion mutants, we demonstrate that the NACHT and LRR domains of NLRP11, but not its pyrin domain, are critical for detection of S. flexneri. Notably, our research reveals that the acid-fast bacterium M. tuberculosis is also detected by NLRP11 and caspase-4, despite not producing LPS. These findings significantly expand the range of pathogens recognized by NLRP11 and caspase-4 to now include acid-fast bacteria that do not contain LPS and underscore the versatility of these innate immune components in pathogen detection.

Autophagy plays a crucial role in the host response to Mycobacterium tuberculosis (Mtb) infection, yet the dynamics and regulation of autophagy induction on Mtb-containing vacuoles (MCVs) remain only partially understood. We employed time-lapse confocal microscopy to investigate the recruitment of LC3B (LC3), a key autophagy marker, to MCVs at the single cell level with our newly developed workflow for single cell and single MCV tracking and fluorescence quantification. We show that approximately 70% of MCVs exhibited LC3 recruitment but that was lost in about 40% of those MCVs. The LC3 recruitment to MCVs displayed a high variability in timing that was independent of the size of the MCV or the bacterial burden. Most notably, the LC3-positive MCVs did not acidify, indicating that LC3 recruitment does not necessarily lead to the formation of mature autophagolysosomes. Interferon-gamma pre-treatment did not affect LC3 recruitment frequency or autophagosome acidification but increased the susceptibility of the macrophage to Mtb-induced cell death. LC3 recruitment and lysotracker staining were mutually exclusive events, alternating on some MCVs multiple times thus demonstrating a reversible aspect of the autophagy response. The LC3 recruitment was associated with galectin-3 and oxysterol-binding protein 1 staining, indicating a correlation with membrane damage and repair mechanisms. ATG7 knock-down did not impact membrane repair, suggesting that autophagy is not directly involved in this process but is coregulated by the membrane damage of MCVs. In summary, our findings provide novel insights into the dynamic and variable nature of LC3 recruitment to the MCVs over time during Mtb infection. Our data does not support a role for autophagy in either cell-autonomous defense against Mtb or membrane repair of the MCV in human macrophages. In addition, the combined dynamics of LC3 recruitment and Lysoview staining emerged as promising markers for investigating the damage and repair processes of phagosomal membranes.

Type I Interferons (IFNs) generally have a protective role during viral infections, but their function during bacterial infections is dependent on the bacterial species. Legionella pneumophila , Shigella sonnei and Mycobacterium tuberculosis can inhibit type I IFN signaling. Here we examined the role of type I IFN, specifically IFNβ, in the context of Salmonella enterica serovar Typhimurium (STm) macrophage infections and the capacity of STm to inhibit type I IFN signaling. We demonstrate that IFNβ has no effect on the intracellular growth of STm in infected bone marrow derived macrophages (BMDMs) derived from C57BL/6 mice. STm infection inhibits IFNβ signaling but not IFNγ signaling in a murine macrophage cell line. We show that this inhibition is independent of the type III and type VI secretion systems expressed by STm and is also independent of bacterial phagocytosis. The inhibition is Toll-like receptor 4 (TLR4)-dependent as the TLR4 ligand, lipopolysaccharide (LPS), alone is sufficient to inhibit IFNβ-mediated signaling and STm-infected, TLR4-deficient BMDMs do not exhibit inhibited IFNβ signaling. In summary, we show that macrophages exposed to STm have reduced IFNβ signaling via crosstalk with TLR4 signaling, and that IFNβ signaling does not affect cell autonomous host defense against STm.

Time-lapse microscopy has emerged as a crucial tool in cell biology, facilitating a deeper understanding of dynamic cellular processes. While existing tracking tools have proven effective in detecting and monitoring objects overtime, the quantification of signals within these tracked objects often faces implementation constraints. In the context of infectious diseases, the quantification of signals at localized compartments within the cell and around intracellular pathogens can provide even deeper insight into the interactions between the pathogen and host cell organelles. Existing quantitative analysis at a single-phagosome level remains limited and dependent on manual tracking methods. We developed a near-fully automated workflow that performs with limited bias, high-throughput cell segmentation and quantitative tracking of both single cell and single bacterium/ phagosome within multi-channel, z-stack, time-lapse confocal microscopy videos. We took advantage of the PyImageJ library to bring Fiji functionality into a Python environment and combined deep- learning-based segmentation from Cellpose with tracking algorithms from Trackmate. The 'da_tracker' workflow provides a versatile toolkit of functions for measuring relevant signal parameters at the singlecell level (such as velocity or bacterial burden) and at the singlephagosome level (i.e. assessment of phagosome maturation over time). Its capabilities in both single-cell and single-phagosome quantification, its flexibility and open-source nature should assist studies that aim to decipher for example the pathogenicity of bacteria and the mechanism of virulence factors that could pave the way for the development of innovative therapeutic approaches.

Accurate quantification of bacterial burden within macrophages, termed Bacterial Burden Quantification (BBQ), is crucial for understanding host-pathogen interactions. Various methods have been employed, each with strengths and weaknesses. This article addresses limitations in existing techniques and introduces two novel automated methods for BBQ within macrophages based on confocal microscopy data analysis. The first method refines total fluorescence quantification by incorporating filtering steps to exclude uninfected cells, while the second method calculates total bacterial volume per cell to mitigate potential biases in fluorescence-based readouts. These workflows utilize PyImageJ and Cellpose software, providing reliable, unbiased, and rapid quantification of bacterial load. The proposed workflows were validated using Salmonella enterica serovar Typhimurium and Mycobacterium tuberculosis models, demonstrating their effectiveness in accurately assessing bacterial burden. These automated workflows offer valuable tools for studying bacterial interactions within host cells and provide insights for various research applications.

Dendritic cells (DCs) are crucial for initiating the acquired immune response to infectious diseases such as tuberculosis. Mycobacterium tuberculosis has evolved strategies to inhibit activation of the NLRP3 inflammasome in macrophages via its serine/threonine protein kinase, protein kinase F (PknF). It is not known whether this pathway is conserved in DCs. In this study, we show that the pknF deletion mutant of M. tuberculosis (MtbΔpknF) compared with wild-type M. tuberculosis-infected cells induces increased production of IL-1β and increased pyroptosis in murine bone marrow-derived DCs (BMDCs). As shown for murine macrophages, the enhanced production of IL-1β postinfection of BMDCs with MtbΔpknF is dependent on NLRP3, ASC, and caspase-1/11. In contrast to macrophages, we show that MtbΔpknF mediates RIPK3/caspase-8-dependent IL-1β production in BMDCs. Consistently, infection with MtbΔpknF results in increased activation of caspase-1 and caspase-8 in BMDCs. When compared with M. tuberculosis-infected cells, the IL-6 production by MtbΔpknF-infected cells was unchanged, indicating that the mutant does not affect the priming phase of inflammasome activation. In contrast, the activation phase was impacted because the MtbΔpknF-induced inflammasome activation in BMDCs depended on potassium efflux, chloride efflux, reactive oxygen species generation, and calcium influx. In conclusion, PknF is important for M. tuberculosis to evade NLRP3 inflammasome-mediated activation of caspase-1 and RIPK3/caspase-8 pathways in BMDCs.

Abstract Translational fidelity is critical for microbial fitness, survival and stress responses. Much remains unknown about the genetic and environmental control of translational fidelity and its single-cell heterogeneity. In this study, we used a high-throughput fluorescence-based assay to screen a knock-out library of Escherichia coli and identified over 20 genes critical for stop-codon readthrough. Most of these identified genes were not previously known to affect translational fidelity. Intriguingly, we show that several genes controlling metabolism, including cyaA and crp, enhance stop-codon readthrough. CyaA catalyzes the synthesis of cyclic adenosine monophosphate (cAMP). Combining RNA sequencing, metabolomics and biochemical analyses, we show that deleting cyaA impairs amino acid catabolism and production of ATP, thus repressing the transcription of rRNAs and tRNAs to decrease readthrough. Single-cell analyses further show that cAMP is a major driver of heterogeneity in stop-codon readthrough and rRNA expression. Our results highlight that carbon metabolism is tightly coupled with stop-codon readthrough.

Pillar[6]MaxQ (P6AS) functions as an in vivo sequestration agent for methamphetamine and fentanyl. We use 1H NMR and isothermal titration calorimetry to glean information on the geometry and strength of the P6AS•drug complexes. P6AS forms tight complexes with fentanyl (Kd = 9.8 nM), PCP (17.1 nM), MDMA (25.5 nM), mephedrone (52.4 nM), and methamphetamine (101 nM). P6AS has good in vitro biocompatibility according to MTS metabolic, Adenylate Kinase cell death, and hERG ion channel inhibition assays, and the Ames fluctuation test. The no observed adverse effect level derived from a maximum tolerated dose study for P6AS was 45 mg kg-1. The hyperlocomotion of mice treated with methamphetamine (0.5 mg kg-1) can be ameliorated by subsequent treatment with P6AS (35.7 mg kg-1) 5-minutes later, whereas the hyperlocomotion of mice treated with fentanyl (0.1 mg kg-1) can be controlled by treatment with P6AS (5 mg kg-1) up to 15-minutes later. P6AS has significant potential for development as a broad spectrum in vivo sequestration agent.

ABSTRACT The host type I interferon (IFN) response protects against Legionella pneumophila infections. Other bacterial pathogens inhibit type I IFN-mediated cell signaling; however, the interaction between this signaling pathway and L. pneumophila has not been well described. Here, we demonstrate that L. pneumophila inhibits the IFN-β signaling pathway but does not inhibit IFN-γ-mediated cell signaling. The addition of IFN-β to L. pneumophila -infected macrophages limited bacterial growth independently of NOS2 and reactive nitrogen species. The type IV secretion system of L. pneumophila is required to inhibit IFN-β-mediated cell signaling. Finally, we show that the inhibition of the IFN-β signaling pathway occurs downstream of STAT1 and STAT2 phosphorylation. In conclusion, our findings describe a novel host cell signaling pathway inhibited by L. pneumophila via its type IV secretion system.

We report studies of the interaction of six acyclic CB[n]-type receptors toward a panel of drugs of abuse by a combination of isothermal titration calorimetry and H-1 NMR spectroscopy. Anthracene walled acyclic CB[n] host (M3) displays highest binding affinity toward methamphetamine (K-d=15 nM) and fentanyl (K-d=4 nM). Host M3 is well tolerated by Hep G2 and HEK 293 cells up to 100 mu M according to MTS metabolic and adenylate kinase release assays. An in vivo maximum tolerated dose study with Swiss Webster mice showed no adverse effects at the highest dose studied (44.7 mg kg(-1)). Host M3 is not mutagenic based on the Ames fluctuation test and does not inhibit the hERG ion channel. In vivo efficacy studies showed that pretreatment of mice with M3 significantly reduces the hyperlocomotion after treatment with methamphetamine, but M3 does not function similarly when administered 30 seconds after methamphetamine.

A recent study in Science found Mycobacterium tuberculosis inhibits pyroptosis of the host cell by secreting a phosphatase (PtpB). PtpB targets the plasma membrane to dephosphorylate PI4P and PI(4,5)P-2, inhibiting recruitment of the pore-forming gasdermin D N-terminal fragment. Pyroptosis inhibition contributes to virulence, as ptpB-deficient Mtb is attenuated in mice.

AbstractAutophagy can act as a defense mechanism for macrophages infected by intracellular pathogens.Mycobacterium tuberculosis(Mtb) is known to both induce and repress autophagic responses, such as xenophagy and LC3-associated phagocytosis (LAP) which both involve the recruitment of LC3 to the Mtb-containing vacuole (MCV). However, the dynamics of MCV interaction with xenophagy or LAP are unclear. Here, using time-lapse confocal microscopy, we present a comprehensive spatio-temporal analysis of the LC3 recruitment to the MCVs during the infection of macrophages. The results revealed frequent LC3 recruitment in the form of large tubule-vesicular structures to the MCV, characteristic of xenophagy, and demonstrated that Mtb could efficiently escape from this signal. We found that the main driver of the LC3 recruitment is the initial macrophage bacterial burden before a second phagocytosis event. We also assessed the potential bactericidal properties of the LC3 recruitment and observed that interferon-gamma treatments did not affect the LC3 recruitment frequency. Additionally, no sign of acidification in the formed autophagosome with or without interferon-gamma treatment was observed. Interestingly, the time-lapses using the acidification probe lysoview revealed that the LC3 recruitment happened shortly after a drop in acidity, a typical sign of membrane damage that is a well-known trigger for autophagy. However, LC3 subsequent loss of signal or escape could also be followed by a restoration of acidification in the vacuole, thus showing restoration of membrane integrity. In conclusion, we show that LC3 recruitment to the MCV correlates with subsequent membrane repair. However, the LC3 recruitment did not show bactericidal properties, questioning its cell intrinsic role in controlling the Mtb infection in macrophages.