Genome-wide screening reveals metabolic regulation of stop-codon readthrough by cyclic AMP.

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.

Legionella pneumophila inhibits type I interferon signaling to avoid cell-intrinsic host cell defense.

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-ß signaling pathway but does not inhibit IFN-γ-mediated cell signaling. The addition of IFN-ß 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-ß-mediated cell signaling. Finally, we show that the inhibition of the IFN-ß 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.

Mycobacterium tuberculosis inhibits the NLRP3 inflammasome activation via its phosphokinase PknF.

Mycobacterium tuberculosis (Mtb) has evolved to evade host innate immunity by interfering with macrophage functions. Interleukin-1ß (IL-1ß) is secreted by macrophages after the activation of the inflammasome complex and is crucial for host defense against Mtb infections. We have previously shown that Mtb is able to inhibit activation of the AIM2 inflammasome and subsequent pyroptosis. Here we show that Mtb is also able to inhibit host cell NLRP3 inflammasome activation and pyroptosis. We identified the serine/threonine kinase PknF as one protein of Mtb involved in the NLRP3 inflammasome inhibition, since the pknF deletion mutant of Mtb induces increased production of IL-1ß in bone marrow-derived macrophages (BMDMs). The increased production of IL-1ß was dependent on NLRP3, the adaptor protein ASC and the protease caspase-1, as revealed by studies performed in gene-deficient BMDMs. Additionally, infection of BMDMs with the pknF deletion mutant resulted in increased pyroptosis, while the IL-6 production remained unchanged compared to Mtb-infected cells, suggesting that the mutant did not affect the priming step of inflammasome activation. In contrast, the activation step was affected since potassium efflux, chloride efflux and the generation of reactive oxygen species played a significant role in inflammasome activation and subsequent pyroptosis mediated by the Mtb pknF mutant strain. In conclusion, we reveal here that the serine/threonine kinase PknF of Mtb plays an important role in innate immune evasion through inhibition of the NLRP3 inflammasome.

In Vitro and In Vivo Sequestration of Phencyclidine by Me4 Cucurbit[8]uril*.

We report investigations of the use of cucurbit[8]uril (CB[8]) macrocycles as an antidote to counteract the in vivo biological effects of phencyclidine. We investigate the binding of CB[8] and its derivative Me4 CB[8] toward ten drugs of abuse (3-9, 12-14) by a combination of 1 H NMR spectroscopy and isothermal titration calorimetry in phosphate buffered water. We find that the cavity of CB[8] and Me4 CB[8] are able to encapsulate the 1-amino-1-aryl-cyclohexane ring system of phencyclidine (PCP) and ketamine as well as the morphinan skeleton of morphine and hydromorphone with Kd values ≤50 nm. In vitro cytotoxicity (MTS metabolic and adenylate kinase cell death assays in HEK293 and HEPG2 cells) and in vivo maximum tolerated dose studies (Swiss Webster mice) which were performed for Me4 CB[8] indicated good tolerability. The tightest host⋅guest pair (Me4 CB[8]⋅PCP; Kd =2 nm) was advanced to in vivo efficacy studies. The results of open field tests demonstrate that pretreatment of mice with Me4 CB[8] prevents subsequent hyperlocomotion induction by PCP and also that treatment of animals previously dosed with PCP with Me4 CB[8] significantly reduces the locomotion levels.

In Vitro and In Vivo Sequestration of Methamphetamine by a Sulfated Acyclic CB[n]-Type Receptor.

We report the synthesis of two new acyclic sulfated acyclic CB[n]-type receptors (TriM0 and Me4 TetM0) and investigations of their binding properties toward a panel of drugs of abuse (1-13) by a combination of 1 H NMR spectroscopy and isothermal titration calorimetry. TetM0 is the most potent receptor with Ka ≥106  M-1 toward methamphetamine, fentanyl, MDMA and mephedrone. TetM0 is not cytotoxic toward HepG2 and HEK 293 cells below 100 µM according to MTS metabolic and adenylate kinase release assays and is well tolerated in vivo when dosed at 46 mg kg-1 . TetM0 does not inhibit the hERG ion channel and is not mutagenic based on the Ames fluctuation test. Finally, in vivo efficacy studies show that the hyperlocomotion of mice treated with methamphetamine can be greatly reduced by treatment with TetM0 up to 5 minutes later. TetM0 has potential as a broad spectrum in vivo sequestrant for drugs of abuse.

Mycobacterium tuberculosis Inhibits Autocrine Type I IFN Signaling to Increase Intracellular Survival.

The type I IFNs (IFN-α and -ß) are important for host defense against viral infections. In contrast, their role in defense against nonviral pathogens is more ambiguous. In this article, we report that IFN-ß signaling in murine bone marrow-derived macrophages has a cell-intrinsic protective capacity against Mycobacterium tuberculosis via the increased production of NO. The antimycobacterial effects of type I IFNs were mediated by direct signaling through the IFN-α/ß-receptor (IFNAR), as Ab-mediated blocking of IFNAR1 prevented the production of NO. Furthermore, M. tuberculosis is able to inhibit IFNAR-mediated cell signaling and the subsequent transcription of 309 IFN-ß-stimulated genes in a dose-dependent way. The molecular mechanism of inhibition by M. tuberculosis involves reduced phosphorylation of the IFNAR-associated protein kinases JAK1 and TYK2, leading to reduced phosphorylation of the downstream targets STAT1 and STAT2. Transwell experiments demonstrated that the M. tuberculosis-mediated inhibition of type I IFN signaling was restricted to infected cells. Overall, our study supports the novel concept that M. tuberculosis evolved to inhibit autocrine type I IFN signaling to evade host defense mechanisms.

Identification of a Transcription Factor That Regulates Host Cell Exit and Virulence of Mycobacterium tuberculosis.

The interaction of Mycobacterium tuberculosis (Mtb) with host cell death signaling pathways is characterized by an initial anti-apoptotic phase followed by a pro-necrotic phase to allow for host cell exit of the bacteria. The bacterial modulators regulating necrosis induction are poorly understood. Here we describe the identification of a transcriptional repressor, Rv3167c responsible for regulating the escape of Mtb from the phagosome. Increased cytosolic localization of MtbΔRv3167c was accompanied by elevated levels of mitochondrial reactive oxygen species and reduced activation of the protein kinase Akt, and these events were critical for the induction of host cell necrosis and macroautophagy. The increase in necrosis led to an increase in bacterial virulence as reflected in higher bacterial burden and reduced survival of mice infected with MtbΔRv3167c. The regulon of Rv3167c thus contains the bacterial mediators involved in escape from the phagosome and host cell necrosis induction, both of which are crucial steps in the intracellular lifecycle and virulence of Mtb.

Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection.

Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the Gα subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens.

Cucurbit[7]uril Enables Multi-Stimuli-Responsive Release from the Self-Assembled Hydrophobic Phase of a Metal Organic Polyhedron.

Mixed self-assembly of ligands 1, 2, 1,6-hexanediamine (HDA), and Pd(NO3)2 afforded Fujita-type metal organic polyhedron MOP1 (diameter ≈ 8.2 nm), which is covalently functionalized with an average of 18 cucurbit[7]uril (CB[7]) units, as evidenced by 1H NMR, diffusion-ordered spectroscopy NMR, and transmission electron microscopy measurements. By virtue of the host-guest properties of CB[7], the inner cavity of MOP can be rendered hydrophobic by using octadecyl HDA (3) as guest during the self-assembly process. The hydrophobic cavity was successfully utilized to trap the hydrophobic dye Nile Red (NR) and the anticancer drug doxorubicin (DOX). The stimuli-responsive release of encapsulated NR or DOX occurs (1) upon addition of a competitive binder (e.g., adamantane ammonium (ADA)) for CB[7], (2) by a dual pH-chemical stimulus involving the protonation state change of adamantane carboxylate at pH 5.8, and (3) by a dual pH-photochemical stimulus involving photoisomerization of trans-6 to cis-6 at pH 5.8. NR is released from NR@MOP2 within HeLa cancer cells. This body of work suggests that the covalent attachment of cucurbit[n]uril to metal organic polyhedra constitutes a promising vehicle for the development of both diagnostic and therapeutic nanoparticles.

Metal-Organic Polyhedron Capped with Cucurbit[8]uril Delivers Doxorubicin to Cancer Cells.

Self-assembly of ligand 1 and Pd(NO3)2 delivers Fujita-type metal-organic polyhedron (MOP) 3 which bears 24 covalently attached methyl viologen units on its external surface, as evidenced by 1H NMR, diffusion-ordered spectroscopy NMR, electrospray mass spectrometry, transmission electron microscopy, and atomic force microscopy measurements. MOP 3 undergoes noncovalent complexation with cucurbit[n]urils to yield MOPs 4-6 with diameter ≈5-6 nm. MOP 5 can be fully loaded with doxorubicin (DOX) prodrug 2 via hetero-ternary complex formation to yield 7. The MOPs exhibit excellent stability toward neutral to slightly acidic pH in 10 mM sodium phosphate buffer, mitigating the concern of disassembly during circulation. The results of MTS assays show that MOP 7 is 10-fold more cytotoxic toward HeLa cells than equimolar quantities of DOX prodrug 2. The enhanced cytotoxicity can be traced to a combination of enhanced cellular uptake of 7 and DOX release as demonstrated by flow cytometry and confocal fluorescence microscopy. The confluence of properties imparted by the polycationic MOP architecture and plug-and-play CB[n] complexation provides a potent new platform for drug delivery application.

Glycoluril-Derived Molecular Clips are Potent and Selective Receptors for Cationic Dyes in Water.

Molecular clip 1 remains monomeric in water and engages in host-guest recognition processes with suitable guests. We report the Ka values for 32 1⋅guest complexes measured by 1 H NMR, UV/Vis, and fluorescence titrations. The cavity of 1 is shaped by aromatic surfaces of negative electrostatic potential and therefore displays high affinity and selectivity for planar and cationic aromatic guests that distinguishes it from CB[n] receptors that prefer aliphatic over aromatic guests. Electrostatic effects play a dominant role in the recognition process whereby ion-dipole interactions may occur between ammonium ions and the C=O groups of 1, between the SO3- groups of 1 and pendant cationic groups on the guest, and within the cavity of 1 by cation-π interactions. Host 1 displays a high affinity toward dicationic guests with large planar aromatic surfaces (e.g. naphthalene diimide NDI+ and perylene diimide PDI+) and cationic dyes derived from acridine (e.g. methylene blue and azure A). The critical importance of cation-π interactions was ascertained by a comparison of analogous neutral and cationic guests (e.g. methylene violet vs. methylene blue; quinoline vs. N-methylquinolinium; acridine vs. N-methylacridinium; neutral red vs. neutral red H+ ) the affinities of which differ by up to 380-fold. We demonstrate that the high affinity of 1 toward methylene blue (Ka =3.92×107 m-1 ; Kd =25 nm) allows for the selective sequestration and destaining of U87 cells stained with methylene blue.

Acyclic Cucurbit[n]uril-Type Molecular Container Enables Systemic Delivery of Effective Doses of Albendazole for Treatment of SK-OV-3 Xenograft Tumors.

Approximately, 40-70% of active pharmaceutical ingredients (API) are severely limited by their extremely poor aqueous solubility, and consequently, there is a high demand for excipients that can be used to formulate clinically relevant doses of these drug candidates. Here, proof-of-concept studies demonstrate the potential of our recently discovered acyclic cucurbit[n]uril-type molecular container Motor1 (M1) as a solubilizing agent for insoluble drugs. M1 did not induce significant rates of mutations in various Salmonella typhimurium test strains during the Ames test, suggesting low genotoxicity. M1 also has low risk of causing cardiac toxicity in humans since it did not inhibit the human Ether-à-go-go-Related Gene channel as tested on transfected CHO cell lines via patch clamp analysis. Albendazole (ABZ) is a widely used antihelminthic agent but that has also shown promising efficacy against cancerous cells in vitro. However, due to its low aqueous solubility (2.7 µM) and poor pharmacokinetics, ABZ is clinically limited as an anticancer agent. Here we investigated the potential of M1 as a solubilizing excipient for ABZ formulation. A pharmacokinetic study indicated that ABZ escapes the peritoneal cavity resulting in 78% absolute bioavailability, while its active intermediate metabolite, albendazole sulfoxide, achieved 43% absolute bioavailability. The daily dosing of 681 mg/kg M1 complexed with 3.2 mg/kg of ABZ for 14 days did not result in significant weight loss or pathology in Swiss Webster mice. In vivo efficacy studies using this M1·ABZ inclusion complex showed significant decreases in tumor growth rates and increases in survival of mice bearing SK-OV-3 xenograft tumors. In conclusion, we provide substantial new evidence demonstrating that M1 is a safe and efficient excipient that enables in vivo parenteral delivery of poorly water-soluble APIs.

A Novel Strategy to Reverse General Anesthesia by Scavenging with the Acyclic Cucurbit[n]uril-type Molecular Container Calabadion 2.

BACKGROUND: Calabadion 2 is a new drug-encapsulating agent. In this study, the authors aim to assess its utility as an agent to reverse general anesthesia with etomidate and ketamine and facilitate recovery. METHODS: To evaluate the effect of calabadion 2 on anesthesia recovery, the authors studied the response of rats to calabadion 2 after continuous and bolus intravenous etomidate or ketamine and bolus intramuscular ketamine administration. The authors measured electroencephalographic predictors of depth of anesthesia (burst suppression ratio and total electroencephalographic power), functional mobility impairment, blood pressure, and toxicity. RESULTS: Calabadion 2 dose-dependently reverses the effects of ketamine and etomidate on electroencephalographic predictors of depth of anesthesia, as well as drug-induced hypotension, and shortens the time to recovery of righting reflex and functional mobility. Calabadion 2 displayed low cytotoxicity in MTS-3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium-based cell viability and adenylate kinase release cell necrosis assays, did not inhibit the human ether-à-go-go-related channel, and was not mutagenic (Ames test). On the basis of maximum tolerable dose and acceleration of righting reflex recovery, the authors calculated the therapeutic index of calabadion 2 in recovery as 16:1 (95% CI, 10 to 26:1) for the reversal of ketamine and 3:1 (95% CI, 2 to 5:1) for the reversal of etomidate. CONCLUSIONS: Calabadion 2 reverses etomidate and ketamine anesthesia in rats by chemical encapsulation at nontoxic concentrations.

A Duplicated ESAT-6 Region of ESX-5 Is Involved in Protein Export and Virulence of Mycobacteria.

The ESX-5 secretion system of Mycobacterium tuberculosis is important for bacterial virulence and for the secretion of the large PE/PPE protein family, whose genes constitute 10% of the M. tuberculosis genome. A four-gene region of the ESX-5 system is duplicated three times in the M. tuberculosis genome, but the functions of these duplicates are unknown. Here we investigated one of these duplicates: the region carrying the esxI, esxJ, ppe15, and pe8 genes (ESX-5a). An ESX-5a deletion mutant in the model system M. marinum background was deficient in the secretion of some members of the PE/PPE family of proteins. Surprisingly, we also identified other proteins that are not members of this family, thus expanding the range of ESX-5 secretion substrates. In addition, we demonstrated that ESX-5a is important for the virulence of M. marinum in the zebrafish model. Furthermore, we showed the role of the M. tuberculosis ESX-5a region in inflammasome activation but not host cell death induction, which is different from the case for the M. tuberculosis ESX-5 system. In conclusion, the ESX-5a region is nonredundant with its ESX-5 paralog and is necessary for secretion of a specific subset of proteins in M. tuberculosis and M. marinum that are important for bacterial virulence of M. marinum. Our findings point to a role for the three ESX-5 duplicate regions in the selection of substrates for secretion via ESX-5, and hence, they provide the basis for a refined model of the molecular mechanism of this type VII secretion system.

Cutting edge: Mycobacterium tuberculosis but not nonvirulent mycobacteria inhibits IFN-ß and AIM2 inflammasome-dependent IL-1ß production via its ESX-1 secretion system.

Mycobacterium tuberculosis extracellular DNA gains access to the host cell cytosol via the ESX-1 secretion system. It is puzzling that this extracellular DNA of M. tuberculosis does not induce activation of the AIM2 inflammasome because AIM2 recognizes cytosolic DNA. In this study, we show that nonvirulent mycobacteria such as Mycobacterium smegmatis induce AIM2 inflammasome activation, which is dependent on their strong induction of IFN-ß production. In contrast, M. tuberculosis, but not an ESX-1-deficient mutant, inhibits the AIM2 inflammasome activation induced by either M. smegmatis or transfected dsDNA. The inhibition does not involve changes in host cell AIM2 mRNA or protein levels but led to decreased activation of caspase-1. We furthermore demonstrate that M. tuberculosis inhibits IFN-ß production and signaling, which was partially responsible for the inhibition of AIM2 activation. In conclusion, we report a novel immune evasion mechanism of M. tuberculosis that involves the ESX-1-dependent, direct or indirect, suppression of the host cell AIM2 inflammasome activation during infection.

BBQ methods: streamlined workflows for bacterial burden quantification in infected cells by confocal microscopy.

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.

da_Tracker: Automated workflow for high throughput single cell and single phagosome tracking in infected cells.

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 over time, 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. Our workflow provides a versatile toolkit of functions for measuring relevant signal parameters at the single-cell level (such as velocity or bacterial burden) and at the single-phagosome level (i.e. assessment of phagosome maturation over time). It's 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.

Salmonella Typhimurium infection inhibits macrophage IFNß signaling in a TLR4-dependent manner.

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 IFNß, 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 IFNß has no effect on the intracellular growth of STm in infected bone marrow derived macrophages (BMDMs) derived from C57BL/6 mice. STm infection inhibits IFNß 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 IFNß-mediated signaling and STm-infected, TLR4-deficient BMDMs do not exhibit inhibited IFNß signaling. In summary, we show that macrophages exposed to STm have reduced IFNß signaling via crosstalk with TLR4 signaling, and that IFNß signaling does not affect cell autonomous host defense against STm.

Mycobacterium tuberculosis genes involved in regulation of host cell death.

The topic of host cell death response upon Mycobacterium tuberculosis (Mtb) infection has been a controversial one [1]. Recent findings demonstrate that one of the important confounding factors was most likely the fact that while Mtb inhibits host cell apoptosis induction early during the infection it clearly induces a necrotic form of cell death during later infection stages [2, 3]. This bi-phasic intracellular lifestyle in regard to host cell death manipulation is emerging as a common theme shared with other facultative and obligate intracellular bacterial pathogens such as Chlamydia and Legionella [4-6]. Accordingly, the list of discovered bacterial proteins involved in host cell apoptosis inhibition is growing [7, 8]. At the same time it is clearly beneficial for the resistance of the host to overcome the bacterial apoptosis block during the early stage of the infection [9-11]. Hence, host cell components have evolved to recognize intracellular pathogens and mediate host cell apoptosis induction if necessary [12]. There have been several reviews on the various aspects of the host cell death response upon Mtb infection [1, 3, 13-15]. Thus in this chapter I will focus on the pathogen side of the equation and describe the tremendous progress that has been made in the identification and characterization of Mtb genes involved in manipulation of host cell death pathways.

BBQ methods: Streamlined workflows for Bacterial Burden Quantification in infected cells by confocal microscopy.

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.