Quantitative Analysis of the Inactivation Process of Internalized Bacteria in Dictyostelium Cells.

The understanding of the inactivation process of ingested bacteria by phagocytes is a key focus in the field of host-pathogen interactions. Dictyostelium is a model organism that has been at the forefront of uncovering the mechanisms underlying this type of interaction. In this study, we describe an assay designed to measure the inactivation of Klebsiella aerogenes in the phagosomes of Dictyostelium discoideum.

Legionella effectors SidC/SdcA ubiquitinate multiple small GTPases and SNARE proteins to promote phagosomal maturation.

Protein ubiquitination is one of the most important posttranslational modifications (PTMs) in eukaryotes and is involved in the regulation of almost all cellular signaling pathways. The intracellular bacterial pathogen Legionella pneumophila translocates at least 26 effectors to hijack host ubiquitination signaling via distinct mechanisms. Among these effectors, SidC/SdcA are novel E3 ubiquitin ligases with the adoption of a Cys-His-Asp catalytic triad. SidC/SdcA are critical for the recruitment of endoplasmic reticulum (ER)-derived vesicles to the Legionella-containing vacuole (LCV). However, the ubiquitination targets of SidC/SdcA are largely unknown, which restricts our understanding of the mechanisms used by these effectors to hijack the vesicle trafficking pathway. Here, we demonstrated that multiple Rab small GTPases and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are bona fide ubiquitination substrates of SidC/SdcA. SidC/SdcA-mediated ubiquitination of syntaxin 3 and syntaxin 4 promotes their unconventional pairing with the vesicle-SNARE protein Sec22b, thereby contributing to the membrane fusion of ER-derived vesicles with the phagosome. In addition, our data reveal that ubiquitination of Rab7 by SidC/SdcA is critical for its association with the LCV membrane. Rab7 ubiquitination could impair its binding with the downstream effector Rab-interacting lysosomal protein (RILP), which partially explains why LCVs avoid fusion with lysosomes despite the acquisition of Rab7. Taken together, our study reveals the biological mechanisms employed by SidC/SdcA to promote the maturation of the LCVs.

The SapM phosphatase can arrest phagosome maturation in an ESX-1 independent manner in Mycobacterium tuberculosis and BCG.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that survives and grows in macrophages. A mechanism used by Mtb to achieve intracellular survival is to secrete effector molecules that arrest the normal process of phagosome maturation. Through phagosome maturation arrest (PMA), Mtb remains in an early phagosome and avoids delivery to degradative phagolysosomes. One PMA effector of Mtb is the secreted SapM phosphatase. Because the host target of SapM, phosphatidylinositol-3-phosphate (PI3P), is located on the cytosolic face of the phagosome, SapM needs to not only be released by the mycobacteria but also travel out of the phagosome to carry out its function. To date, the only mechanism known for Mtb molecules to leave the phagosome is phagosome permeabilization by the ESX-1 secretion system. To understand this step of SapM function in PMA, we generated identical in-frame sapM mutants in both the attenuated Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine strain, which lacks the ESX-1 system, and Mtb. Characterization of these mutants demonstrated that SapM is required for PMA in BCG and Mtb. Further, by establishing a role for SapM in PMA in BCG, and subsequently in a Mtb mutant lacking the ESX-1 system, we demonstrated that the role of SapM does not require ESX-1. We further determined that ESX-2 or ESX-4 is also not required for SapM to function in PMA. These results indicate that SapM is a secreted effector of PMA in both BCG and Mtb, and that it can function independent of the known mechanism for Mtb molecules to leave the phagosome.

Profiling phagosome proteins identifies PD-L1 as a fungal-binding receptor.

Phagocytosis is the process by which myeloid phagocytes bind to and internalize potentially dangerous microorganisms1. During phagocytosis, innate immune receptors and associated signalling proteins are localized to the maturing phagosome compartment, forming an immune information processing hub brimming with microorganism-sensing features2-8. Here we developed proximity labelling of phagosomal contents (PhagoPL) to identify proteins localizing to phagosomes containing model yeast and bacteria. By comparing the protein composition of phagosomes containing evolutionarily and biochemically distinct microorganisms, we unexpectedly identified programmed death-ligand 1 (PD-L1) as a protein that specifically enriches in phagosomes containing yeast. We found that PD-L1 directly binds to yeast upon processing in phagosomes. By surface display library screening, we identified the ribosomal protein Rpl20b as a fungal protein ligand for PD-L1. Using an auxin-inducible depletion system, we found that detection of Rpl20b by macrophages cross-regulates production of distinct cytokines including interleukin-10 (IL-10) induced by the activation of other innate immune receptors. Thus, this study establishes PhagoPL as a useful approach to quantifying the collection of proteins enriched in phagosomes during host-microorganism interactions, exemplified by identifying PD-L1 as a receptor that binds to fungi.

Legionella pneumophila exploits the endo-lysosomal network for phagosome biogenesis by co-opting SUMOylated Rab7.

Legionella pneumophila strains harboring wild-type rpsL such as Lp02rpsLWT cannot replicate in mouse bone marrow-derived macrophages (BMDMs) due to induction of extensive lysosome damage and apoptosis. The bacterial factor directly responsible for inducing such cell death and the host factor involved in initiating the signaling cascade that leads to lysosome damage remain unknown. Similarly, host factors that may alleviate cell death induced by these bacterial strains have not yet been investigated. Using a genome-wide CRISPR/Cas9 screening, we identified Hmg20a and Nol9 as host factors important for restricting strain Lp02rpsLWT in BMDMs. Depletion of Hmg20a protects macrophages from infection-induced lysosomal damage and apoptosis, allowing productive bacterial replication. The restriction imposed by Hmg20a was mediated by repressing the expression of several endo-lysosomal proteins, including the small GTPase Rab7. We found that SUMOylated Rab7 is recruited to the bacterial phagosome via SulF, a Dot/Icm effector that harbors a SUMO-interacting motif (SIM). Moreover, overexpression of Rab7 rescues intracellular growth of strain Lp02rpsLWT in BMDMs. Our results establish that L. pneumophila exploits the lysosomal network for the biogenesis of its phagosome in BMDMs.

Group A Streptococcus induces lysosomal dysfunction in THP-1 macrophages.

The human-specific bacterial pathogen group A Streptococcus (GAS) is a significant cause of morbidity and mortality. Macrophages are important to control GAS infection, but previous data indicate that GAS can persist in macrophages. In this study, we detail the molecular mechanisms by which GAS survives in THP-1 macrophages. Our fluorescence microscopy studies demonstrate that GAS is readily phagocytosed by macrophages, but persists within phagolysosomes. These phagolysosomes are not acidified, which is in agreement with our findings that GAS cannot survive in low pH environments. We find that the secreted pore-forming toxin Streptolysin O (SLO) perforates the phagolysosomal membrane, allowing leakage of not only protons but also large proteins including the lysosomal protease cathepsin B. Additionally, GAS recruits CD63/LAMP-3, which may contribute to lysosomal permeabilization, especially in the absence of SLO. Thus, although GAS does not inhibit fusion of the lysosome with the phagosome, it has multiple mechanisms to prevent proper phagolysosome function, allowing for persistence of the bacteria within the macrophage. This has important implications for not only the initial response but also the overall functionality of the macrophages, which may lead to the resulting pathologies in GAS infection. Our data suggest that therapies aimed at improving macrophage function may positively impact patient outcomes in GAS infection.

Staphylococcus aureus promotes its intracellular survival by inhibiting Rab11-Rab11FIP4-mediated vesicle trafficking.

Mastitis in dairy cows is mainly caused by bacteria, in which Staphylococcus aureus appears frequently. Epithelial cells, as a major physical barrier of mammary gland, play an important role in preventing mastitis in dairy cows. Our previous study reported that Rab11fip4 (an effector of Rab11) was significantly changed in response to stimulation by S. aureus. So, in this study, the role of Rab11A in phagocytosis of bovine mammary epithelial cells (MAC-T) against S. aureus was evaluated. First, changes of Rab11A and Rab11fip4 were analyzed in response to S. aureus by immunofluorescence and western blotting. Subsequently, the effects of Rab11A and Rab11fip4 on proliferation of S. aureus, as well as formation and function of late endosomes (LEs) and lysosomes (LYSs) were investigated. The results showed that, after infection, Rab11A and Rab11fip4 were recruited to phagosomes containing S. aureus. Rab11A promoted bacterial clearance and rescues the destruction of LEs and LYSs by S. aureus, whereas Rab11fip4 did the opposite. These findings provide new insights into phagocytosis and control of S. aureus in host cells, thus lay the foundation to elucidate the pathogenesis of S. aureus in bovine mastitis.

Mycobacterium tuberculosis hijacks host macrophages-derived interleukin 16 to block phagolysosome maturation for enhancing intracellular growth.

The discovery of promising cytokines and clarification of their immunological mechanisms in controlling the intracellular fate of Mycobacterium tuberculosis (Mtb) are necessary to identify effective diagnostic biomarkers and therapeutic targets. To escape immune clearance, Mtb can manipulate and inhibit the normal host process of phagosome maturation. Phagosome maturation arrest by Mtb involves multiple effectors and much remains unknown about this important aspect of Mtb pathogenesis. In this study, we found that interleukin 16 (IL-16) is elevated in the serum samples of Tuberculosis (TB) patients and can serve as a specific target for treatment TB. There was a significant difference in IL-16 levels among active TB, latent TB infection (LTBI), and non-TB patients. This study first revealed that macrophages are the major source of IL-16 production in response to Mtb infection, and elucidated that IL-16 can promote Mtb intracellular survival by inhibiting phagosome maturation and suppressing the expression of Rev-erbα which can inhibit IL-10 secretion. The experiments using zebrafish larvae infected with M. marinum and mice challenged with H37Rv demonstrated that reducing IL-16 levels resulted in less severe pathology and improved survival, respectively. In conclusion, this study provided direct evidence that Mtb hijacks the host macrophages-derived interleukin 16 to enhance intracellular growth. It is suggesting the immunosuppressive role of IL-16 during Mtb infection, supporting IL-16 as a promising therapeutic target.

4-(Benzyloxy)phenol-induced p53 exhibits antimycobacterial response triggering phagosome-lysosome fusion through ROS-dependent intracellular Ca2+ pathway in THP-1 cells.

Drug-resistant tuberculosis (TB) outbreak has emerged as a global public health crisis. Therefore, new and innovative therapeutic options like host-directed therapies (HDTs) through novel modulators are urgently required to overcome the challenges associated with TB. In the present study, we have investigated the anti-mycobacterial effect of 4-(Benzyloxy)phenol. Cell-viability assay asserted that 50 µM of 4-(Benzyloxy)phenol was not cytotoxic to phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 (dTHP-1) cells. It was observed that 4-(Benzyloxy)phenol activates p53 expression by hindering its association with KDM1A. Increased ROS, intracellular Ca2+ and phagosome-lysosome fusion, were also observed upon 4-(Benzyloxy)phenol treatment. 4-(Benzyloxy)phenol mediated killing of intracellular mycobacteria was abrogated in the presence of specific inhibitors of ROS, Ca2+ and phagosome-lysosome fusion like NAC, BAPTA-AM, and W7, respectively. We further demonstrate that 4-(Benzyloxy)phenol mediated enhanced ROS production is mediated by acetylation of p53. Blocking of p53 acetylation by Pifithrin-α (PFT- α) enhanced intracellular mycobacterial growth by blocking the mycobactericidal effect of 4-(Benzyloxy)phenol. Altogether, the results showed that 4-(Benzyloxy)phenol executed its anti-mycobacterial effect by modulating p53-mediated ROS production to regulate phagosome-lysosome fusion through Ca2+ production.

Pathogenicity and virulence of Francisella tularensis.

Tularaemia is a zoonotic disease caused by the Gram-negative bacterium, Francisella tularensis. Depending on its entry route into the organism, F. tularensis causes different diseases, ranging from life-threatening pneumonia to less severe ulceroglandular tularaemia. Various strains with different geographical distributions exhibit different levels of virulence. F. tularensis is an intracellular bacterium that replicates primarily in the cytosol of the phagocytes. The main virulence attribute of F. tularensis is the type 6 secretion system (T6SS) and its effectors that promote escape from the phagosome. In addition, F. tularensis has evolved a peculiar envelope that allows it to escape detection by the immune system. In this review, we cover tularaemia, different Francisella strains, and their pathogenicity. We particularly emphasize the intracellular life cycle, associated virulence factors, and metabolic adaptations. Finally, we present how F. tularensis largely escapes immune detection to be one of the most infectious and lethal bacterial pathogens.

Host-directed therapy for bacterial infections -Modulation of the phagolysosome pathway.

Bacterial infections still impose a significant burden on humanity, even though antimicrobial agents have long since been developed. In addition to individual severe infections, the f fatality rate of sepsis remains high, and the threat of antimicrobial-resistant bacteria grows with time, putting us at inferiority. Although tremendous resources have been devoted to the development of antimicrobial agents, we have yet to recover from the lost ground we have been driven into. Looking back at the evolution of treatment for cancer, which, like infectious diseases, has the similarity that host immunity eliminates the lesion, the development of drugs to eliminate the tumor itself has shifted from a single-minded focus on drug development to the establishment of a treatment strategy in which the de-suppression of host immunity is another pillar of treatment. In infectious diseases, on the other hand, the development of therapies that strengthen and support the immune system has only just begun. Among innate immunity, the first line of defense that bacteria encounter after invading the host, the molecular mechanisms of the phagolysosome pathway, which begins with phagocytosis to fusion with lysosome, have been elucidated in detail. Bacteria have a large number of strategies to escape and survive the pathway. Although the full picture is still unfathomable, the molecular mechanisms have been elucidated for some of them, providing sufficient clues for intervention. In this article, we review the host defense mechanisms and bacterial evasion mechanisms and discuss the possibility of host-directed therapy for bacterial infection by intervening in the phagolysosome pathway.

Subversion strategies of lysosomal killing by intracellular pathogens.

Many pathogenic organisms need to reach either an intracellular compartment or the cytoplasm of a target cell for their survival, replication or immune system evasion. Intracellular pathogens frequently penetrate into the cell through the endocytic and phagocytic pathways (clathrin-mediated endocytosis, phagocytosis and macropinocytosis) that culminates in fusion with lysosomes. However, several mechanisms are triggered by pathogenic microorganisms - protozoan, bacteria, virus and fungus - to avoid destruction by lysosome fusion, such as rupture of the phagosome and thereby release into the cytoplasm, avoidance of autophagy, delaying in both phagolysosome biogenesis and phagosomal maturation and survival/replication inside the phagolysosome. Here we reviewed the main data dealing with phagosome maturation and evasion from lysosomal killing by different bacteria, protozoa, fungi and virus.

Activation of inflammasomes and mechanisms for intracellular recognition of Listeria monocytogenes.

The high mortality rate associated with Listeria monocytogenes can be attributed to its ability to invade the body systemically and to activate inflammasomes. Both of these processes are facilitated by expressing a major virulence factor known as listeriolysin O, a 56 kDa pore-forming protein encoded by the hly gene. Listeriolysin O plays a crucial role in the pathogenesis of the bacterium by facilitating the escape of the pathogen from the phagosome into the cytosol. This process is essential for the successful establishment of infection. In addition, listeriolysin O is known as an immunomodulator that activates host signal transduction. In addition to listeriolysin O, Listeria expresses a variety of bacterial ligands, such as lipoteichoic acid, nucleotide, and flagellin, that are recognized by host intracellular pattern-recognition receptors including Nod-like receptors, AIM2-like receptors, and RIG-I-like receptors. This review introduces intracellular recognition of Listeria monocytogenes since recent studies have revealed that the activation of inflammasome exacerbates Gram-positive bacteria infection.

The nucleotide receptor STING translocates to the phagosomes to negatively regulate anti-fungal immunity.

STING (stimulator of interferon genes) exerts protective cellular responses to viral infection via induction of interferon production and autophagy. Here, we report the role of STING in modulating the immune responses toward fungal infection. Upon Candida albicans stimulation, STING transited alongside the endoplasmic reticulum (ER) to the phagosomes. In phagosomes, STING directly bound with Src via the N-terminal 18 amino acids of STING, and this binding prevented Src from recruiting and phosphorylating Syk. Consistently, Syk-associated signaling and production of pro-inflammatory cytokines and chemokines were increased in mouse BMDCs (bone-marrow-derived dendritic cells) lacking STING with fungal treatment. STING deficiency improved anti-fungal immunity in systemic C. albicans infection. Importantly, administration of the N-terminal 18-aa (amino acid) peptide of STING improved host outcomes in disseminated fungal infection. Overall, our study identifies a previously unrecognized function of STING in negatively regulating anti-fungal immune responses and offers a potential therapeutic strategy for controlling C. albicans infection.

The dynamics of Mycobacterium tuberculosis phagosome and the fate of infection.

Phagosomes are vesicles produced by phagocytosis of phagocytes, which are crucial in immunity against Mycobacterium tuberculosis (Mtb) infection. After the phagocyte ingests the pathogen, it activates the phagosomes to recruit a series of components and process proteins, to phagocytose, degrade and kill Mtb. Meanwhile, Mtb can resist acid and oxidative stress, block phagosome maturation, and manipulate host immune response. The interaction between Mtb and phagocytes leads to the outcome of infection. The dynamic of this process can affect the cell fate. This article mainly reviews the development and maturation of phagosomes, as well as the dynamics and modifications of Mtb effectors and phagosomes components, and new diagnostic and therapeutic markers involved in phagosomes.

Histoplasma capsulatum Relies on Tryptophan Biosynthesis To Proliferate within the Macrophage Phagosome.

Histoplasma capsulatum yeasts reside and proliferate within the macrophage phagosome during infection. This nutrient-depleted phagosomal environment imposes challenges to Histoplasma yeasts for nutrition acquisition. Histoplasma yeasts require all 20 amino acids, which can be formed by de novo biosynthesis and/or acquired directly from the phagosomal environment. We investigated how Histoplasma obtains aromatic amino acids (i.e., phenylalanine, tyrosine, and tryptophan) within the phagosome during infection of macrophages. Depletion of key enzymes of the phenylalanine or tyrosine biosynthetic pathway neither impaired Histoplasma's ability to proliferate within macrophages nor resulted in attenuated virulence in vivo. However, loss of tryptophan biosynthesis resulted in reduced growth within macrophages and severely attenuated virulence in vivo. Together, these results indicate that phenylalanine and tyrosine, but not tryptophan, are available to Histoplasma within the macrophage phagosome. The herbicide glyphosate, which targets 5-enolpyruvylshikimate-3-phosphate synthase of the aromatic amino acid biosynthetic pathway, inhibited Histoplasma yeast growth, and this growth inhibition was partially reversed by aromatic amino acid supplementation or overexpression of ARO1. These results suggest that the aromatic amino acid biosynthetic pathway is a candidate drug target to develop novel antifungal therapeutics.

Inside the phagosome: A bacterial perspective.

The neutrophil phagosome is one of the most hostile environments that bacteria must face and overcome if they are to succeed as pathogens. Targeting bacterial defense mechanisms should lead to new therapies that assist neutrophils to kill pathogens, but this has not yet come to fruition. One of the limiting factors in this effort has been our incomplete knowledge of the complex biochemistry that occurs within the rapidly changing environment of the phagosome. The same compartmentalization that protects host tissue also limits our ability to measure events within the phagosome. In this review, we highlight the limitations in our knowledge, and how the contribution of bacteria to the phagosomal environment is often ignored. There appears to be significant heterogeneity among phagosomes, and it is important to determine whether survivors have more efficient defenses or whether they are ingested into less threatening environments than other bacteria. As part of these efforts, we discuss how monitoring or recovering bacteria from phagosomes can provide insight into the conditions they have faced. We also encourage the use of unbiased screening approaches to identify bacterial genes that are essential for survival inside neutrophil phagosomes.

High content quantitative imaging of Mycobacterium tuberculosis responses to acidic microenvironments within human macrophages.

Intracellular pathogens such as Mycobacterium tuberculosis (Mtb) have evolved diverse strategies to counteract macrophage defence mechanisms including phagolysosomal biogenesis. Within macrophages, Mtb initially resides inside membrane-bound phagosomes that interact with lysosomes and become acidified. The ability of Mtb to control and subvert the fusion between phagosomes and lysosomes plays a key role in the pathogenesis of tuberculosis. Therefore, understanding how pathogens interact with the endolysosomal network and cope with intracellular acidification is important to better understand the disease. Here, we describe in detail the use of fluorescence microscopy-based approaches to investigate Mtb responses to acidic environments in cellulo. We report high-content imaging modalities to probe Mtb sensing of external pH or visualise in real-time Mtb intrabacterial pH within infected human macrophages. We discuss various methodologies with step-by-step analyses that enable robust image-based quantifications. Finally, we highlight the advantages and limitations of these different approaches and discuss potential alternatives that can be applied to further investigate Mtb-host cell interactions. These methods can be adapted to study host-pathogen interactions in different biological systems and experimental settings. Altogether, these approaches represent a valuable tool to further broaden our understanding of the cellular and molecular mechanisms underlying intracellular pathogen survival.

Zooming in on common immune evasion mechanisms of pathogens in phagolysosomes: potential broad-spectrum therapeutic targets against infectious diseases.

The intracellular viral, bacterial, or parasitic pathogens evade the host immune challenges to propagate and cause fatal diseases. The microbes overpower host immunity at various levels including during entry into host cells, phagosome formation, phagosome maturation, phagosome-lysosome fusion forming phagolysosomes, acidification of phagolysosomes, and at times after escape into the cytosol. Phagolysosome is the final organelle in the phagocyte with sophisticated mechanisms to degrade the pathogens. The immune evasion strategies by the pathogens include the arrest of host cell apoptosis, decrease in reactive oxygen species, the elevation of Th2 anti-inflammatory response, avoidance of autophagy and antigen cross-presentation pathways, and escape from phagolysosomal killing. Since the phagolysosome organelle in relation to infection/cure is seldom discussed in the literature, we summarize here the common host as well as pathogen targets manipulated or utilized by the pathogens established in phagosomes and phagolysosomes, to hijack the host immune system for their benefit. These common molecules or pathways can be broad-spectrum therapeutic targets for drug development for intervention against infectious diseases caused by different intracellular pathogens.

Persistence of Mycobacterium tuberculosis in response to infection burden and host-induced stressors.

Introduction: As infection with Mycobacterium tuberculosis progresses, the bacilli experience various degrees of host stressors in the macrophage phagosome such as low pH, nutrient deprivation, or exposure to toxic agents, which promotes cell-to-cell phenotypic variation. This includes a physiologically viable but non- or slowly replicating persister subpopulation, which is characterised by a loss of growth on solid media, while remaining metabolically active. Persisters additionally evade the host immune response and macrophage antimicrobial processes by adapting their metabolic pathways to maintain survival and persistence in the host. Methods: A flow cytometry-based dual-fluorescent replication reporter assay, termed fluorescence dilution, provided a culture-independent method to characterize the single-cell replication dynamics of M. tuberculosis persisters following macrophage infection. Fluorescence dilution in combination with reference counting beads and a metabolic esterase reactive probe, calcein violet AM, provided an effective approach to enumerate and characterize the phenotypic heterogeneity within M. tuberculosis following macrophage infection. Results: Persister formation appeared dependent on the initial infection burden and intracellular bacterial burden. However, inhibition of phagocytosis by cytochalasin D treatment resulted in a significantly higher median percentage of persisters compared to inhibition of phagosome acidification by bafilomycin A1 treatment. Discussion: Our results suggest that different host factors differentially impact the intracellular bacterial burden, adaptive mechanisms and entry into persistence in macrophages.