Professional Phagocytes Research Articles

Neutrophils enhance the clearance of systemic amyloid deposits in a murine amyloidoma model

IntroductionAmyloid-specific antibodies have been shown to opsonize and enhance amyloid clearance in systemic amyloidosis mouse models. However, the immunological mechanisms by which amyloid is removed have not been clearly defined. Previous reports from preclinical in vivo studies suggest polymorphonuclear cells (i.e., neutrophils) can affect amyloid removal. Therefore, we sought to analyze how neutrophils may contribute to the clearance of human AL amyloid extracts, using a murine amyloidoma model.MethodsImmunocompromised nude mice injected subcutaneously with patient-derived AL amyloid extract (generating a localized “amyloidoma”) were used to circumvent confounding factors contributed by the adaptive immune system and served as the model system. Two representative AL amyloid extracts were used, ALλ(CLA), which is refractory to clearance, and ALκ(TAL), which is readily cleared in mice. Neutrophil recruitment to the amyloid masses, cellular activation, and propensity to engulf amyloid were assessed.ResultsImmunophenotyping of amyloidomas from animals implanted with 2 mg of either ALλ or ALκ revealed that more neutrophils were recruited to ALκ amyloid masses as compared to the ALλ material, which was generally devoid of neutrophils. Ex vivo analyses indicated neutrophils do not efficiently phagocytose amyloid directly. However, histological evaluation of the ALκ amyloidoma revealed the abundant presence of neutrophil extracellular traps, which were absent in the ALλ amyloidomas. Using neutrophil depletion experiments in mice, we determined that mice devoid of neutrophils cleared the human amyloid lesions less efficiently. Moreover, mice devoid of neutrophils also had significantly reduced intra-amyloid expression of inflammatory cytokines.DiscussionNeutrophils may not directly mediate amyloid clearance through phagocytosis; however, these cells can be stimulated by the amyloid and may function to facilitate phagocytosis and amyloid clearance by professional phagocytes (e.g., macrophages).

A bottom-up approach identifies the antipsychotic and antineoplastic trifluoperazine and the ribose derivative deoxytubercidin as novel microglial phagocytosis inhibitors.

Phagocytosis is an indispensable function of microglia, the brain professional phagocytes. Microglia is particularly efficient phagocytosing cells that undergo programmed cell death (apoptosis) in physiological conditions. However, mounting evidence suggests microglial phagocytosis dysfunction in multiple brain disorders. These observations prompted us to search for phagocytosis modulators (enhancers or inhibitors) with therapeutic potential. We used a bottom-up strategy that consisted on the identification of phagocytosis modulators using phenotypic high throughput screenings (HTSs) in cell culture and validation in organotypic cultures and in vivo. We performed two complementary HTS campagnes: at Achucarro, we used primary cultures of mouse microglia and compounds of the Prestwick Chemical Library; at Roche, we used human iPSC derived macrophage-like cells and a proprietary chemo-genomic library with 2200 compounds with known mechanism-of-action. Next, we validated the more robust compounds using hippocampal organotypic cultures and identified two phagocytosis inhibitors: trifluoperazine, a dopaminergic and adrenergic antagonist used as an antipsychotic and antineoplastic; and deoxytubercidin, a ribose derivative. Finally, we tested whether these compounds were able to modulate phagocytosis of apoptotic newborn cells in the adult hippocampal neurogenic niche in vivo by administering them into the mouse hippocampus using osmotic minipumps. We confirmed that both trifluoperazine and deoxytubercidin have anti-phagocytic activity in vivo, and validated our bottom-up strategy to identify novel phagocytosis modulators. These results show that chemical libraries with annotated mechanism of action are an starting point for the pharmacological modulation of microglia in drug discovery projects aiming at the therapeutic manipulation of phagocytosis in brain diseases.

Stem cells tightly regulate dead cell clearance to maintain tissue fitness

Billions of cells are eliminated daily from our bodies1–4. Although macrophages and dendritic cells are dedicated to migrating and engulfing dying cells and debris, many epithelial and mesenchymal tissue cells can digest nearby apoptotic corpses1–4. How these non-motile, non-professional phagocytes sense and eliminate dying cells while maintaining their normal tissue functions is unclear. Here we explore the mechanisms that underlie their multifunctionality by exploiting the cyclical bouts of tissue regeneration and degeneration during hair cycling. We show that hair follicle stem cells transiently unleash phagocytosis at the correct time and place through local molecular triggers that depend on both lipids released by neighbouring apoptotic corpses and retinoids released by healthy counterparts. We trace the heart of this dual ligand requirement to RARγ–RXRα, whose activation enables tight regulation of apoptotic cell clearance genes and provides an effective, tunable mechanism to offset phagocytic duties against the primary stem cell function of preserving tissue integrity during homeostasis. Finally, we provide functional evidence that hair follicle stem cell-mediated phagocytosis is not simply redundant with professional phagocytes but rather has clear benefits to tissue fitness. Our findings have broad implications for other non-motile tissue stem or progenitor cells that encounter cell death in an immune-privileged niche.

Manipulation of Macrophages: Emerging Mechanisms of Leishmaniasis.

As professional phagocytes, macrophages represent the first line of defence against invading microbial pathogens. Various cellular processes such as programmed cell death, autophagy and RNA interference (RNAi) of macrophages are involved directly in elimination or assist in elimination of invading pathogens. However, parasites, such as Leishmania, have evolved diverse strategies to interfere with macrophage cell functions, favouring their survival, growth and replication inside hostile and restrictive environments of macrophages. Therefore, identification and detailed characterization of macrophage-pathogen interactions is the key to understanding how pathogens subvert macrophage functions to support their infection and disease process. In recent years, great progress has been achieved in understanding how Leishmania affects with critical host macrophage functions. Based on latest progress and accumulating knowledge, this review exclusively focuses on macrophage-Leishmania interaction, providing an overview of macrophage cellular processes such as programmed cell death, autophagy and RNAi during Leishmania infection. Despite extensive progress, many questions remain and require further investigation.

Signaling by Neutrophil G Protein-Coupled Receptors that Regulate the Release of Superoxide Anions.

In human peripheral blood, the neutrophil granulocytes (neutrophils) are the most-abundant white blood cells. These professional phagocytes are rapidly recruited from the bloodstream to inflamed tissues by chemotactic factors that signal danger. Neutrophils, which express many receptors that are members of the large family of G protein-coupled receptors (GPCRs), are critical for the elimination of pathogens and inflammatory insults, as well as for the resolution of inflammation leading to tissue repair. Danger-signaling molecular patterns such as the N-formylated peptides that are formed during bacterial and mitochondrial protein synthesis and recognized by formyl peptide receptors (FPRs) and free fatty acids recognized by free fatty acid receptors (FFARs) regulate neutrophil functions. Short peptides and short chain fatty acids activate FPR1 and FFA2R, respectively, while longer peptides and fatty acids activate FPR2 and GPR84, respectively. The activation profiles of these receptors include the release of reactive oxygen species (ROS) generated by the NADPH oxidase. Activation of the oxidase and the production of ROS are processes that are regulated by proinflammatory mediators, including TNFα and GM-CSF. The receptors have signaling and functional similarities, although there are also important differences, not only between the two closely related neutrophil FPRs, but also between the FPRs and the FFARs. In neutrophils, these receptors never walk alone, and additional mechanistic insights into the regulation of the GPCRs and the novel regulatory mechanisms underlying the activation of NADPH oxidase advance our understanding of the role of receptor transactivation in the regulation of inflammatory reactions.

Plasma membrane abundance dictates phagocytic capacity and functional cross-talk in myeloid cells.

Professional phagocytes like neutrophils and macrophages tightly control what they consume, how much they consume, and when they move after cargo uptake. We show that plasma membrane abundance is a key arbiter of these cellular behaviors. Neutrophils and macrophages lacking the G protein subunit Gβ4 exhibited profound plasma membrane expansion, accompanied by marked reduction in plasma membrane tension. These biophysical changes promoted the phagocytosis of bacteria, fungus, apoptotic corpses, and cancer cells. We also found that Gβ4-deficient neutrophils are defective in the normal inhibition of migration following cargo uptake. Sphingolipid synthesis played a central role in these phenotypes by driving plasma membrane accumulation in cells lacking Gβ4. In Gβ4 knockout mice, neutrophils not only exhibited enhanced phagocytosis of inhaled fungal conidia in the lung but also increased trafficking of engulfed pathogens to other organs. Together, these results reveal an unexpected, biophysical control mechanism central to myeloid functional decision-making.

Peripheral endotoxin exposure in mice activates crosstalk between phagocytes in the brain and periphery.

Inflammation is a central process of many neurological diseases, and a growing number of studies suggest that non-brain-resident immune cells may contribute to this neuroinflammation. However, the unique contributions of specific immune cell subsets to neuroinflammation are presently unknown, and it is unclear how communication between brain-resident and non-resident immune cells underlies peripheral immune cell involvement in neuroinflammation. In this study, we employed the well-established model of lipopolysaccharide (LPS)-induced neuroinflammation and captured brain-resident and non-resident immune cells from the brain and its vasculature by magnetically enriching cell suspensions from the non-perfused brain for CD45 + cells. Then, we identified immune subtype-specific neuroinflammatory processes using single-cell genomics and predicted the crosstalk between immune cell subtypes by analyzing the simultaneous expression of ligands and receptors. We observed a greater abundance of peripheral phagocytes associated with the brain in this model of neuroinflammation, and report that these professional phagocytes activated similar transcriptional profiles to microglia during LPS-induced neuroinflammation. And, we observed that the probable crosstalk between microglia and peripheral phagocytes was activated in this model while homotypic microglial communication was likely to be decreased. Our novel findings reveal that microglia signaling to non-brain-resident peripheral phagocytes is preferentially triggered by peripheral inflammation, which is associated with brain infiltration of peripheral cells. Overall, our study supports the involvement of peripheral immune cells in neuroinflammation and suggests several possible molecular signaling pathways between microglia and peripheral cells that may facilitate central-peripheral crosstalk during inflammation. Examining these molecular mediators in human disease and other rodent models may reveal novel targets that modify brain health, especially in comorbidities characterized by peripheral inflammation.

Repeated trans-spinal magnetic stimulation promotes microglial phagocytosis of myelin debris after spinal cord injury through LRP-1

Spinal cord injury (SCI) is a serious trauma of the central nervous system. The clearance of myelin debris is a critical step in the functional recovery following spinal cord injury (SCI). Recent studies have begun to reveal critical roles for professional phagocytes in the central nervous system, microglia, and their receptors in the control of myelin debris in neurodegenerative disease. Repeated trans-spinal magnetic stimulation (rTSMS) has been demonstrated as a noninvasive SCI treatment that enhances tissue repair and functional recovery. In this study, we investigated the role and molecular mechanism of rTSMS on microglial phagocytosis of myelin debris in a rat SCI model. In our studies, we found that rTSMS significantly promoted the motor function recovery of SCI rats associated with the inhibition the neuroinflammation and glia scar formation. Immunofluorescence results further showed that the rTSMS promotes the clearance of myelin debris by microglia in vivo and in vitro. Additionally, receptor-associated protein (RAP), a Low-density lipoprotein receptor-related protein-1 (LRP-1) inhibitor, could cancel the accelerated microglial phagocytosis of myelin debris after rTSMS in vitro experiments. Simultaneously, Elisa's results and western blotting respectively showed that rTSMS significantly decreased the levels of soluble LRP-1(sLRP-1) and the LRP-1 splicing enzyme of ADAM17. In conclusion, rTSMS could promote the clearance of myelin debris by microglia through LRP-1 to improve the functional recovery of SCI rats.

Engineering Efferocytosis-Mimicking Nanovesicles to Regulate Joint Anti-Inflammation and Peripheral Immunosuppression for Rheumatoid Arthritis Therapy.

Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic inflammation of the synovial joints and the dysfunction of regulatory T cells (Tregs) in the peripheral blood. Therefore, an optimal treatment strategy should aim to eliminate the inflammatory response in the joints and simultaneously restore the immune tolerance of Tregs in peripheral blood. Accordingly, we developed an efferocytosis-mimicking nanovesicle that contains three functional factors for immunomodulating of efferocytosis, including "find me" and "eat me" signals for professional (macrophage) or non-professional phagocytes (T lymphocyte), and "apoptotic metabolite" for metabolite digestion. We showed that efferocytosis-mimicking nanovesicles targeted the inflamed joints and spleen of mice with collagen-induced arthritis, further recruiting and selectively binding to macrophages and T lymphocytes to induce M2 macrophage polarization and Treg differentiation and T helper cell 17 (Th17) recession. Under systemic administration, the efferocytosis-mimicking nanovesicles effectively maintained the pro-inflammatory M1/anti-inflammatory M2 macrophage balance in joints and the Treg/Th17 imbalance in peripheral blood to prevent RA progression. This study demonstrates the potential of efferocytosis-mimicking nanovesicles for RA immunotherapy.

Glioblastoma Phagocytic Cell Death: Balancing the Opportunities for Therapeutic Manipulation.

Macrophages and microglia are professional phagocytes that sense and migrate toward "eat-me" signals. The role of phagocytic cells is to maintain homeostasis by engulfing senescent or apoptotic cells, debris, and abnormally aggregated macromolecules. Usually, dying cells send out "find-me" signals, facilitating the recruitment of phagocytes. Healthy cells can also promote or inhibit the phagocytosis phenomenon of macrophages and microglia by tuning the balance between "eat-me" and "don't-eat-me" signals at different stages in their lifespan, while the "don't-eat-me" signals are often hijacked by tumor cells as a mechanism of immune evasion. Using a combination of bioinformatic analysis and spatial profiling, we delineate the balance of the "don't-eat-me" CD47/SIRPα and "eat-me" CALR/STC1 ligand-receptor interactions to guide therapeutic strategies that are being developed for glioblastoma sequestered in the central nervous system (CNS).

Efferocytosis by macrophages in physiological and pathological conditions: regulatory pathways and molecular mechanisms.

Efferocytosis is defined as the highly effective phagocytic removal of apoptotic cells (ACs) by professional or non-professional phagocytes. Tissue-resident professional phagocytes ("efferocytes"), such as macrophages, have high phagocytic capacity and are crucial to resolve inflammation and aid in homeostasis. Recently, numerous exciting discoveries have revealed divergent (and even diametrically opposite) findings regarding metabolic immune reprogramming associated with efferocytosis by macrophages. In this review, we highlight the key metabolites involved in the three phases of efferocytosis and immune reprogramming of macrophages under physiological and pathological conditions. The next decade is expected to yield further breakthroughs in the regulatory pathways and molecular mechanisms connecting immunological outcomes to metabolic cues as well as avenues for "personalized" therapeutic intervention.

TAG-FREE GLYCOSYLATED RHMFG-E8 AS A THERAPY FOR ACUTE KIDNEY INJURY.

Background: Acute kidney injury (AKI) can result from renal ischemia and reperfusion (I/R) and often occurs during surgical procedures in cardiac, liver, kidney transplantation, and trauma-hemorrhage. Milk fat globule epidermal growth factor-factor VIII (MFG-E8) functions as a bridging molecule to promote the removal of dying cells by professional phagocytes. Because MFG-E8 promotes clearance of apoptotic cells, we have explored its therapeutic potential in various organ injury conditions. To develop human MFG-E8 as a potential therapy, we have generated a human cell-expressed, and thus glycosylated, tag-free recombinant human (rh) MFG-E8 and tested its safety and biological activity in vitro . We hypothesize that the tag-free glycosylated rhMFG-E8 is protective in I/R-induced AKI and it can be developed as an effective therapy for AKI. Methods: To assess the pharmacokinetic properties of the tag-free rhMFG-E8, Sprague-Dawley rats were either untreated or treated with a bolus dose of the tag-free rhMFG-E8, blood collected at various time points and the recovery of human MFG-E8 in the blood were measured by ELISA. Adult male C57BL6 mice underwent bilateral renal ischemia for 30 min, and immediately upon reperfusion, mice were treated intraperitoneally with either normal saline (vehicle) or 20 μg/kg human cell expressed, glycosylated tag-free rhMFG-E8. At either 24 h or 48 h after I/R, blood and kidneys were harvested for further analysis. In separate cohorts of mice after I/R and treatment, mice were observed for 10 days, and survival recorded. Results: AKI rats treated with the tag-free rhMFG-E8 had similar half-life as those in the treated control rats. At 48 h after I/R-induced AKI, renal function markers, blood urea nitrogen, and creatinine were increased and treatment with the tag-free rhMFG-E8 significantly decreased these markers. At both 24 h and 48 h after AKI, inflammatory cytokines, TNF-α, IL-6, and IL-1β were increased and treatment decreased these levels. The kidney mRNA expressions of these cytokines were also increased at 24 h after AKI and treatment significantly decreased those mRNA expressions. Histologically, at 48 h after AKI, tubular damage, and the number of TUNEL staining cells were increased and treatment markedly decreased these measurements. Administration of tag-free rhMFG-E8 at the time of reperfusion improved survival in a 10-day survival study. Conclusion: Our new human cell-expressed tag-free rhMFG-E8 is protective in I/R-induced AKI and it may have the potential to be further developed as a safe and effective therapy for AKI.

Deciphering Staphylococcus aureus-host dynamics using dual activity-based protein profiling of ATP-interacting proteins.

This study uses a chemoproteomic approach to target active ATP-interacting proteins and examines the dynamic proteomic interactions between Staphylococcus aureus and human cell lines THP-1 and HaCaT. It uncovers the distinct responses of macrophages and keratinocytes during bacterial infection. S. aureus demonstrated a tailored response to the intracellular environment of each cell type and adaptation during exposure to professional and non-professional phagocytes. It also highlights strategies employed by S. aureus to persist within host cells. This study offers significant insights into the human cell response to S. aureus infection, illuminating the complex proteomic shifts that underlie the defense mechanisms of macrophages and keratinocytes. Notably, the study underscores the nuanced interplay between the host's metabolic reprogramming and immune strategy, suggesting potential therapeutic targets for enhancing host defense and inhibiting bacterial survival. The findings enhance our understanding of host-pathogen interactions and can inform the development of targeted therapies against S. aureus infections.

Interferons: Invited guests at the brain’s gala banquet

Removal of toxic debris that can hinder brain function is performed primarily by microglia, the brain's professional phagocytes. A recent study in Cell1 identified that viral response interferons are required for priming microglia, ensuring competent phagocytosis and proper circuit wiring.

IL-12 Mediates T-bet-Expressing Myeloid Cell-Dependent Host Resistance against Toxoplasma gondii.

To defend against intracellular pathogens such as Toxoplasma gondii, the host generates a robust type 1 immune response. Specifically, host defense against T. gondii is defined by an IL-12-dependent IFN-γ response that is critical for host resistance. Previously, we demonstrated that host resistance is mediated by T-bet-dependent ILC-derived IFN-γ by maintaining IRF8+ conventional type 1 dendritic cells during parasitic infection. Therefore, we hypothesized that innate lymphoid cells are indispensable for host survival. Surprisingly, we observed that T-bet-deficient mice succumb to infection quicker than do mice lacking lymphocytes, suggesting an unknown T-bet-dependent-mediated host defense pathway. Analysis of parasite-mediated inflammatory myeloid cells revealed a novel subpopulation of T-bet+ myeloid cells (TMCs). Our results reveal that TMCs have the largest intracellular parasite burden compared with other professional phagocytes, suggesting they are associated with active killing of T. gondii. Mechanistically, we established that IL-12 is necessary for the induction of inflammatory TMCs during infection and these cells are linked to a role in host survival.

1,25 dihydroxyvitamin D3-mediated effects on bovine innate immunity and on biofilm-forming Staphylococcus spp. isolated from cattle with mastitis

Mastitis is one the most widespread and serious diseases in dairy cattle. Recurrent and chronic infections are often attributable to certain pathogenicity mechanisms in mastitis-causing pathogens such as Staphylococcus spp. These include growing in biofilm and invading cells, both of which make it possible to resist or evade antimicrobial therapies and the host’s immune system. This study tested the effects of active vitamin D3 (i.e., calcitriol or 1,25-dihydroxyvitamin D3) on the internalization and phagocytosis of biofilm-forming Staphylococcus spp. isolated from animals with mastitis. Two established bovine cell lines were used: MAC-T (mammary epithelial cells) and BoMac (macrophages). Calcitriol (0–200 nM) did not affect the viability of MAC-T cells nor that of BoMac cells after 24 and 72 h. Concentrations of 0–100 mM for 24 h upregulated the expression of 24-hydroxylase in MAC-T cells, but did not alter that of VDR. Pre-treatment of the cells with calcitriol for 24 h decreased the internalization of S. aureus V329 into MAC-T cells (0–100 nM), and stimulated the phagocytosis of the same strain and of S. xylosus 4913 (0–10 nM). Calcitriol and two conditioned media, obtained by treating the cells with 25–200 nM of the metabolite for 24 h, were also assessed in terms of their antimicrobial and antibiofilm activity. Neither calcitriol by itself nor the conditioned media affected staphylococcal growth or biofilm formation (0–200 nM for 12 and 24 h, respectively). In contrast, the conditioned media (0–100 nM for 24 h) decreased the biomass of preformed non-aureus staphylococcal biofilms and killed the bacteria within them, without affecting metabolic activity. These effects may be mediated by reactive oxygen species and proteins with antimicrobial and/or antibiofilm activity. In short, calcitriol could make pathogens more accessible to antimicrobial therapies and enhance bacterial clearance by professional phagocytes. Moreover, it may modulate the host’s endogenous defenses in the bovine udder and help combat preformed non-aureus staphylococcal biofilms (S. chromogenes 40, S. xylosus 4913, and/or S. haemolyticus 6). The findings confirm calcitriol’s potential as an adjuvant to prevent and/or treat intramammary infections caused by Staphylococcus spp., which would in turn contribute to reducing antibiotic use on dairy farms.

Stabilin-2 mediated apoptotic cell phagocytosis induces interleukin-10 expression by p38 and Pbx1 signaling

Apoptotic cell death occurs under normal physiological conditions, such as development, tissue remodeling, and inflammation. Appropriate removal of apoptotic cells by phagocytes and the secretion of anti-inflammatory cytokines such as IL-10 are important mechanisms for maintaining tissue homeostasis. Apoptotic cell phagocytosis is mediated by several phosphatidylserine recognition receptors on non-professional or professional phagocytes, such as neighboring epithelial cells or macrophages. Stabilin-2 is reported as a phosphatidylserine recognition receptor for apoptotic cell phagocytosis, and its downstream signaling pathway for cytoskeletal rearrangement for phagocytosis is well known. However, the mechanisms for stabilin-2-mediated IL-10 production has not yet been reported. In this study, we aimed to investigate stabilin-2 receptor-mediated IL-10 transcription regulation signaling pathway.

Brucella infection and Toll-like receptors.

Brucella consists of gram-negative bacteria that have the ability to invade and replicate in professional and non-professional phagocytes, and its prolonged persistence in the host leads to brucellosis, a serious zoonosis. Toll-like receptors (TLRs) are the best-known sensors of microorganisms implicated in the regulation of innate and adaptive immunity. In particular, TLRs are transmembrane proteins with a typical structure of an extracellular leucine-rich repeat (LRR) region and an intracellular Toll/interleukin-1 receptor (TIR) domain. In this review, we discuss Brucella infection and the aspects of host immune responses induced by pathogens. Furthermore, we summarize the roles of TLRs in Brucella infection, with substantial emphasis on the molecular insights into its mechanisms of action.

Quantification of Polystyrene Uptake by Different Cell Lines Using Fluorescence Microscopy and Label-Free Visualization of Intracellular Polystyrene Particles by Raman Microspectroscopic Imaging.

Environmental pollution caused by plastic is a present problem. Polystyrene is a widely used packaging material (e.g., Styrofoam) that can be broken down into microplastics through abrasion. Once the plastic is released into the environment, it is dispersed by wind and atmospheric dust. In this study, we investigated the uptake of polystyrene particles into human cells using A549 cells as a model of the alveolar epithelial barrier, CaCo-2 cells as a model of the intestinal epithelial barrier, and THP-1 cells as a model of immune cells to simulate a possible uptake of microplastics by inhalation, oral uptake, and interaction with the cellular immune system, respectively. The uptake of fluorescence-labeled beads by the different cell types was investigated by confocal laser scanning microscopy in a semi-quantitative, concentration-dependent manner. Additionally, we used Raman spectroscopy as a complementary method for label-free qualitative detection and the visualization of polystyrene within cells. The uptake of polystyrene beads by all investigated cell types was detected, while the uptake behavior of professional phagocytes (THP-1) differed from that of adherent epithelial cells.

Phylogenomic, structural, and cell biological analyses reveal that Stenotrophomonas maltophilia replicates in acidified Rab7A-positive vacuoles of Acanthamoeba castellanii.

Acanthamoeba species are clinically relevant free-living amoebae (FLA) ubiquitously found in soil and water bodies. Metabolically active trophozoites graze on diverse microbes via phagocytosis. However, functional studies on Rab GTPases (Rabs), which are critical for controlling vesicle trafficking and maturation, are scarce for this FLA. This knowledge gap can be partly explained by the limited genetic tools available for Acanthamoeba cell biology. Here, we developed plasmids to generate fusions of A. castellanii strain Neff proteins to the N- or C-termini of mEGFP and mCherry2. Phylogenomic and structural analyses of the 11 Neff Rab7 paralogs found in the RefSeq assembly revealed that eight of them had non-canonical sequences. After correcting the gene annotation for the Rab7A ortholog, we generated a line stably expressing an mEGFP-Rab7A fusion, demonstrating its correct localization to acidified macropinocytic and phagocytic vacuoles using fluorescence microscopy live cell imaging (LCI). Direct labeling of live Stenotrophomonas maltophilia ESTM1D_MKCAZ16_6a (Sm18) cells with pHrodo Red, a pH-sensitive dye, demonstrated that they reside within acidified, Rab7A-positive vacuoles. We constructed new mini-Tn7 delivery plasmids and tagged Sm18 with constitutively expressed mScarlet-I. Co-culture experiments of Neff trophozoites with Sm18::mTn7TC1_Pc_mScarlet-I, coupled with LCI and microplate reader assays, demonstrated that Sm18 underwent multiple replication rounds before reaching the extracellular medium via non-lytic exocytosis. We conclude that S. maltophilia belongs to the class of bacteria that can use amoeba as an intracellular replication niche within a Stenotrophomonas-containing vacuole that interacts extensively with the endocytic pathway.IMPORTANCEDiverse Acanthamoeba lineages (genotypes) are of increasing clinical concern, mainly causing amoebic keratitis and granulomatous amebic encephalitis among other infections. S. maltophilia ranks among the top 10 most prevalent multidrug-resistant opportunistic nosocomial pathogens and is a recurrent member of the microbiome hosted by Acanthamoeba and other free-living amoebae. However, little is known about the molecular strategies deployed by Stenotrophomonas for an intracellular lifestyle in amoebae and other professional phagocytes such as macrophages, which allow the bacterium to evade the immune system and the action of antibiotics. Our plasmids and easy-to-use microtiter plate co-culture assays should facilitate investigations into the cellular microbiology of Acanthamoeba interactions with Stenotrophomonas and other opportunistic pathogens, which may ultimately lead to the discovery of new molecular targets and antimicrobial therapies to combat difficult-to-treat infections caused by these ubiquitous microbes.