A Genome-Wide Screen in Macrophages Defines Host Genes Regulating the Uptake of Mycobacterium abscessus

ABSTRACT The interactions between a host cell and a pathogen can dictate disease outcomes and are important targets for host-directed therapies. Mycobacterium abscessus (Mab) is a highly antibiotic resistant, rapidly growing nontuberculous mycobacterium that infects patients with chronic lung diseases. Mab can infect host immune cells, such as macrophages, which contribute to its pathogenesis. However, our understanding of initial host-Mab interactions remains unclear. Here, we developed a functional genetic approach to define these host-Mab interactions by coupling a Mab fluorescent reporter with a genome-wide knockout library in murine macrophages. We used this approach to conduct a forward genetic screen to define host genes that contribute to the uptake of Mab by macrophages. We identified known regulators of phagocytosis, such as the integrin ITGB2, and uncovered a key requirement for glycosaminoglycan (sGAG) synthesis for macrophages to efficiently take up Mab. CRISPR-Cas9 targeting of three key sGAG biosynthesis regulators, Ugdh, B3gat3, and B4galt7 resulted in reduced uptake of both smooth and rough Mab variants by macrophages. Mechanistic studies suggest that sGAGs function upstream of pathogen engulfment and are required for the uptake of Mab, but not Escherichia coli or latex beads. Further investigation found that the loss of sGAGs reduced the surface expression, but not the mRNA expression, of key integrins, suggesting an important role for sGAGs in modulating surface receptor availability. Together, these studies globally define and characterize important regulators of macrophage-Mab interactions and are a first step to understanding host genes that contribute to Mab pathogenesis and disease. IMPORTANCE Pathogen interactions with immune cells like macrophages contribute to pathogenesis, yet the mechanisms underlying these interactions remain largely undefined. For emerging respiratory pathogens, like Mycobacterium abscessus, understanding these host-pathogen interactions is important to fully understand disease progression. Given that M. abscessus is broadly recalcitrant to antibiotic treatments, new therapeutic approaches are needed. Here, we leveraged a genome-wide knockout library in murine macrophages to globally define host genes required for M. abscessus uptake. We identified new macrophage uptake regulators during M. abscessus infection, including a subset of integrins and the glycosaminoglycan synthesis (sGAG) pathway. While ionic characteristics of sGAGs are known to drive pathogen-cell interactions, we discovered a previously unrecognized requirement for sGAGs to maintain robust surface expression of key uptake receptors. Thus, we developed a flexible forward-genetic pipeline to define important interactions during M. abscessus infection and more broadly identified a new mechanism by which sGAGs control pathogen uptake.

is the second most common NTM respiratory pathogen recovered in the United States, accounting for a significant number of rapidly growing mycobacterial respiratory disease isolates (3). Due to its recalcitrance to many antibiotics, current treatment success rates remain below 50% (4)(5)(6). Treatment is further complicated by the ability of Mab to transition from a smooth to rough morphology that is driven by mutations that alter the export of the glycolipoproteins (GPLs) to the surface of Mab, including mutations in the transporter MmpL4 (7,8). This transition to the rough variant increases biofilm formation and decreases antibiotic sensitivity (9). Thus, Mab is an emerging pathogen of clinical importance and there is a critical need to develop new treatment options.
Developing more effective therapies requires a deeper understanding of Mab pathogenesis and host-pathogen interactions that drive infection. Recent work suggests that Mab interactions with macrophages are critical for disease progression, including the transition of smooth Mab variants into rough variants (3,(10)(11)(12). Both smooth and rough Mab variants can survive in macrophages with rough variants initiating more rapid cell death cascades (3,10,11). Transposon mutagenesis studies and other genetic approaches in Mab have identified many genes that are required for effective antibiotic killing and intracellular survival, including the ESX-IV system (13)(14)(15). This virulence determinant contributes to the inhibition of lysosomal fusion enabling intracellular survival of Mab in macrophages (15). In contrast to what is known about Mab, little is known about macrophage genes required for Mab uptake and survival. While studies have shown that Mab interacts with TLR2 and Dectin-1 to initiate uptake, a systematic characterization of Mab uptake by macrophages remains lacking (16,17).
Uptake of pathogens requires effective interactions between the pathogen and macrophages. Macrophages use surface pattern recognition receptors (PRRs), including toll-like receptors, mannose receptors and scavenger receptors, for the initial recognition of many pathogens (18)(19)(20)(21)(22). Other modifications to surface proteins, including sulfated glycosaminoglycan (sGAG) modifications such as heparin sulfate and chondroitin sulfate, affect the efficiency of pathogen attachment to the cell surface (23,24). On epithelial cells, sGAGs play an important role for the attachment of several pathogens, including Coronavirus and Chlamydia trachomatis (25)(26)(27). It is predicted that these surface modifications alter the charge interactions between pathogens and the host cell surface to modulated initial attachment (28,29).
Following adherence, phagocytosis or receptor mediated engulfment of pathogens enables internalization. While each phagocytic cargo has unique components, the general pathway for phagocytosis is conserved. Phagocytosis begins with the generation of a phagosome following a ligand binding to macrophage surface receptors (30). This binding event activates downstream signaling cascades that remodel the actin cytoskeleton to drive the progressive engagement of additional receptors around the particle (31,32). After engulfment, controlled membrane fusion events result in the formation of the nascent phagosome. The phagosome then interacts with various endosome components that acidify the compartment and ultimately lead to compartment fusion with the lysosome (33)(34)(35)(36)(37)(38). Several elegant studies have dissected unique pathways that contribute to phagocytosis of distinct cargos, defining receptors and signaling cascades that are essential (39)(40)(41). However, no studies have globally examined host genes that control Mab interactions with macrophages.
Here, we defined the host genes that control the uptake of Mab into macrophages by coupling a brightly fluorescent Mab reporter with a genome-wide CRISPR-Cas9-mediated macrophage knockout library in murine immortalized bone marrow-derived macrophages (iBMDMs). We performed a forward genetic screen to enrich for macrophages that could or could not take up Mab identifying several previously defined phagocytosis components. Follow-up studies uncovered a strong requirement for sGAG production in macrophages for efficient Mab uptake. While loss of sGAGs did not affect general phagocytosis pathways or opsonized uptake of Mab, sGAGs were essential for initial interactions of both smooth and rough Mab variants with macrophages. Mechanistic studies uncovered a role for sGAGs in maintaining high surface expression of the key integrins ITGB2 and ITGAL, suggesting a new role for sGAGs in maintaining receptor availability on the cell surface. These results uncover important host pathways that modulate the interactions between Mab and macrophages during infection.

RESULTS
Fluorescent Mab reporter enables dissection of macrophage-Mab interactions. While Mab infects macrophages, our understanding of these interactions remains limited. To help address this gap in knowledge we developed a brightly fluorescent Mab reporter strain. A constitutive mEmerald GFP vector was transformed into the smooth variant of Mab strain ATCC-19977. Comparisons to nonfluorescent Mab showed that bacterial growth and antibiotic mediated killing with rifampicin in liquid media were unaffected by fluorescent protein expression (Fig. 1A). We next examined the utility of the fluorescent Mab strain to dissect host-pathogen interactions with macrophages. We first examined the robustness of the fluorescent reporter by infecting immortalized bone marrow derived macrophages (iBMDMs) from C57BL/6J mice. One previous study using a GFP fluorescent Mab was unable to detect increased fluorescence in macrophages until 24 h of infection, when attachment, uptake and bacterial growth all contribute to differential fluorescence (42). We hypothesized that earlier time points could be examined using the brighter mEmerald GFP, allowing us to distinguish uptake from intracellular growth. To test this hypothesis, we quantified the number of mEmerald positive macrophages by flow cytometry 6 h after infecting iBMDMs with fluorescent Mab at increasing multiplicities of infection (MOIs). We noted that increasing the MOI resulted in an increase in the percentage of mEmerald1 cells and the mean fluorescence intensity of these infected cells (Fig. 1B to 1D). We next quantified the mEmerald1 cells over the first 6 h of infection of iBMDMs at an MOI of 5. We observed the number of infected cells 2-h postinfection showed a minimal increase compared to uninfected controls, while there was a significant increase in infected cells at four-and 6-h postinfection ( Fig. 1E and F). Thus, the mEmerald GFP reporter does not affect Mab growth while enabling the dissection of early interactions between Mab and macrophages.
CRISPR-Cas9 loss-of-function (LOF) screen identifies host genes required for Mab uptake by macrophages. Host pathways required for macrophage uptake of Mab remain almost entirely unknown. To test if the flow cytometry-based uptake assay could identify host pathways required for Mab uptake, we used a known phagocytosis inhibitor, Cytochalasin D, to inhibit actin polymerization (43). iBMDMs were pretreated with Cytochalasin D then infected with mEmerald Mab. The percentage of cells that were infected were then quantified 4 h later by flow cytometry. We observed a nearly 50% decrease in mEmerald1 macrophages treated with Cytochalasin D compared to vehicle controls ( Fig. 2A and B). These data highlight the sensitivity of the uptake assay to dissect early interactions between Mab and macrophages.
Next, we leveraged the uptake assay to globally identify host regulators required for Mab uptake by macrophages (Fig. 2C). We previously generated a robust and reproducible pooled genome-wide loss-of-function library in Cas91 iBMDMs (44). Knockouts in this pool were generated with sgRNAs from the Brie library which targets four independent single guide RNA molecules (sgRNAs) per coding gene and over 1,000 nontargeting controls (NTC) (45). To identify regulators of Mab uptake, we infected the loss-of-function library of macrophages with mEmerald Mab for 4 h. Given our goal to identify host genes that specifically modulate Mab uptake, we chose to complete this screen at 4 h postinfection since this is the earliest time point with a significant increase of mEmerald that is detectable by flow cytometry. Fluorescence activated cells sorting (FACS) was then used to isolate over 200Â sgRNA coverage of the library from mEmerald1 cells and mEmerald-cells. Following genomic DNA extraction, sgRNA abundances for each sorted bin were quantified by deep sequencing.
To test for statistical enrichment of sgRNAs and genes, we used the modified robust rank algorithm (a-RRA) employed by Model-based Analysis of Genome-wide CRISPR/ Cas9 Knockout (MAGeCK). This algorithm ranks sgRNAs by effect before filtering low ranking sgRNAs to improve significance testing. To identify macrophage genes required for Mab uptake during early infection, we compared the enrichment of sgRNAs in the mEmerald -(Mab uninfected) directly to the mEmerald1 (Mab infected) population (Table S1). The a-RRA analysis identified 100 genes with a P value , 0.01 and a fold change of 2 with at least 2 independent sgRNAs. Among the top 100 candidates, we identified known regulators of macrophage phagocytosis, including Manea, M6PR, Itgb2  (46). Guide-level analysis showed agreement with all four sgRNAs targeting these genes suggesting they are bona fide hits and that our screen was robust (Table S1).
To confirm the screen results, we validated the role of Manea, M6PR, Itgb2, and Itgam in controlling Mab uptake by macrophages. We generated two independent iBMDM lines targeting two distinct sgRNAs per gene, in addition to a nontargeting control. These cells were then infected with mEmerald Mab and uptake was quantified 4 h later. In agreement with our screen results, we found that targeting each candidate gene resulted in a significant decrease in mEmerald1 iBMDMs compared to NTC cells (Fig. 2E). Taken together these results show that the genome-wide screen identified host genes that contribute to early interactions between Mab and macrophages.
Sulfated glycosaminoglycans (sGAGs) are required for macrophage uptake of Mab. To discover new pathways that are required for Mab uptake by macrophages, we next used DAVID analysis and gene set enrichment analysis (GSEA) to identify functional enrichments from our uptake screen data set (Table S2). These analyses identified the sulfated glycosaminoglycan (sGAG) synthesis pathway as required for efficient uptake of Mab ( Fig. 3A and Table S2). This leading-edge analysis showed a clear enrichment of 12 sGAG-biosynthesis genes in the uninfected population (left side enrichment) compared to Mab infected cells. While a variety of pathogens use sGAGs to facilitate attachment and invasion of epithelial cells, little is known about their role in macrophages. Within the top candidates in the screen were multiple genes within the sGAG biosynthesis pathway (Fig. 3B). Three of these genes, UGDH, B3GAT3, and B4GALT7 encode UDP-glucose 6dehydrogenase, galactosyltransferase I and glucuronosyltransferase I, respectively, and were among the top 30 candidates while showing strong agreement among the four independent sgRNAs (Table S1). These data suggest that sGAGs contribute to Mab uptake by macrophages.
To validate the importance of macrophage sGAGs for Mab uptake, we targeted Ugdh, B3gat3, and B4galt7 directly in iBMDMs. Using an approach targeting two independent sgRNAs simultaneously we generated a panel of iBMDMs with editing efficiency ranging from 60 to 95% in each gene, and one cell line per gene was selected for follow up studies (45). We first examined functional differences following gene editing by quantifying the total sGAGs in sGAG-targeted and NTC iBMDMs. We found that iBMDMs targeted for Ugdh, B3gat3, and B4galt7 each resulted in a significant reduction in sGAGs compared to NTC macrophages (Fig. 3C). We next used these sGAG-targeted cells to examine differences in Mab uptake. NTC and sGAG-targeted iBMDMs were infected with mEmerald Mab for 4 h with increasing MOIs and bacterial uptake was quantified. At each MOI we observed a significant decrease in Mab uptake in sGAG-targeted iBMDMs compared to the NTC cells ( Fig. 3D and 3E). These results confirm that sGAGs contribute to the effective uptake of Mab by macrophages, validating our genome-wide screen and bioinformatic analysis.
sGAGs are required for macrophage uptake of rough Mab variants. While our data suggest sGAGs are needed for uptake of smooth Mab variants by macrophages, it remained unclear if this host pathway was also required for rough variant uptake. To directly test this question, a rough Mab variant derived from ATCC 19977 was transformed with the mEmerald reporter. Similar to the smooth variant, we noted no defects in growth or antibiotic killing in broth culture (Fig. 4A) and robust uptake by iBMDMs in an MOI dependent manner (Fig. 4B to D). To test if sGAGs are also required for uptake of rough Mab we next infected NTC and sGAG-targeted iBMDMs with the rough mEmerald Mab reporter for 4 h at increasing MOI and quantified the percentage of infected cells. We observed over a 50% decrease of rough Mab uptake in sGAG-targeted macrophages compared to control macrophages at all MOIs ( Fig. 4E and F). Interestingly, while significant, the magnitude of the decrease in uptake in sGAG-targeted iBMDMs was lower for rough Mab than smooth Mab. Taken together these data show sGAGs are required for macrophage uptake of both smooth and rough Mab variants.
sGAGs function upstream of Mab internalization. We next examined the mechanisms underlying sGAG-mediated control of Mab uptake. To understand if sGAGs control specific or general uptake pathways, we tested whether sGAGs were necessary for uptake of other phagocytic cargo by macrophages. First, we quantified the uptake of latex beads by incubating yellow-green-labeled beads at increasing concentrations with control or sGAG-targeted iBMDMs. In contrast to our results with Mab, we found no differences in the uptake of latex beads between control or sGAG-targeted iBMDMs ( Fig. 5A and B). We next examined whether sGAGs contribute to the uptake of Escherichia coli, a Gram-negative bacterium. mEmerald expressing E. coli was incubated with NTC or sGAGtargeted iBMDMs for 4 h then uptake was quantified. Similar to latex beads, we observed no significant differences in E. coli uptake between control or sGAG-targeted macrophages ( Fig. 5C and D). Taken together, these results suggest that sGAG-mediated  Given that the uptake of pathogens can be influenced by opsonization, we next examined whether the sGAG pathway overlaps with complement-mediated uptake mechanisms. Both smooth and rough mEmerald Mab reporters were incubated in active or heat killed serum for 30 min. Following serum incubation, control or sGAG-targeted iBMDMs were infected and the percent uptake of Mab was quantified 4 h later. Opsonization of the smooth Mab variant with active serum resulted in no significant change in uptake compared to heat-inactivated serum (Fig. 5E). For the rough Mab variant, while uptake was significantly increased following incubation with active serum in all cells, the uptake differences between NTC and sGAG-targeted iBMDMs remained significant (Fig. 5F). These data show that while smooth and rough Mab variants are differentially susceptible to complement-mediated phagocytosis, this uptake pathway is independent of sGAGs.
To distinguish if sGAGs are required for internalization or attachment of Mab we next treated control or sGAG-targeted macrophages with Cytochalasin D. We hypothesized that if sGAGs control initial attachment that blocking actin polymerization with Cytochalasin D would further inhibit Mab uptake in sGAG-targeted macrophages. To test this hypothesis, we pretreated NTC and sGAG-targeted macrophages with DMSO or Cytochalasin D for 2 h. Cells were then infected with smooth or rough variants of mEmerald Mab and uptake was quantified 4 h later. We found that Cytochalasin D treatment significantly reduced uptake of both smooth and rough Mab variants compared to vehicle controls in all cell lines tested ( Fig. 5G and H). These data suggest that sGAGs are needed upstream of bacterial internalization as they function additively with actin polymerization inhibitors.
Loss of sGAGs reduces the surface integrin expression on macrophages. While sGAGs directly modulate ionic interactions at the cellular surface, it remained possible that sGAG-modifications also regulate the surface levels of the receptors that are required for pathogen uptake (29). Given the importance of integrins for Mab uptake from our screen, we examined the expression of a subset of integrins, including ITGB2, ITGAM, and ITGAL. When we quantified the mRNA levels of these integrins we found no changes in the mRNA expression of integrins between NTC and sGAG-targeted macrophages (Fig. 6A). In contrast, while we observed no difference in the surface expression of ITGAM, we observed a significant decrease in ITGB2 and ITGAL expression on the surface of sGAG-targeted iBMDMs ( Fig. 6B and C). We next directly examined surface levels of the ITGB2/ITGAL heterodimer (LFA-1) and found significantly reduced expression on the surface of sGAG-targeted iBMDMs ( Fig. 6D and E). Taken together these data suggest that sGAGs modulate the surface expression of key integrins that contribute to Mab uptake by macrophages.

DISCUSSION
Infections with M. abscessus are on the rise globally and their recalcitrance to antibiotic therapy is driving treatment failure (47)(48)(49). Designing effective treatments against Mab, including host-directed therapies, will require a fundamental understanding of critical host-Mab interactions that occur during infection. Here, we developed a Mab fluorescent reporter-based approach to investigate interactions of Mab with macrophages and conducted a forward genetic screen to globally identify host genes that are required for Mab uptake. Our screen identified many candidates, including previously described players in pathogen uptake such as Itgb2, Itgam, Manea, and M6pr (50, 51), which together with our validation highlight the robustness of this data set and the strengths of the functional genetic approach.
As part of our bioinformatic analysis to uncover new pathways required for efficient Mab uptake, we identified a strong signature for the sGAG biosynthesis pathway. sGAGs are modified glycosaminoglycans, which consist of repeating polysaccharides attached to the cell surface and surface proteins (29). We targeted three independent genes in the sGAG synthesis pathway that all resulted in decreased uptake of both smooth and rough Mab variants by macrophages. While sGAGs have previously been shown to modulate host-pathogen interactions in epithelial cells, very little is known about their role in macrophages (25)(26)(27). We found that sGAGs did not affect latex bead or E. coli uptake, showing that general macrophage uptake mechanisms are intact in sGAG-targeted cells. These findings are in line with a report that used iterative CRISPR screens to identify genes needed for the phagocytosis of distinct cargo, including beads of distinct sizes and complement/IgG coated cargo (40). This wide-ranging study did not identify a significant enrichment of sGAG biosynthetic genes in any of their screens suggesting further specificity of the sGAG-mediated uptake pathway. In addition, Cytochalasin D treatment of sGAG-targeted macrophages further reduced Mab uptake, suggesting sGAGs contribute upstream of the actin polymerization required for Mab internalization. Together these data point toward a role for sGAGs in the adherence of Mab to macrophages. While many models suggest sGAGs modulate ionic interactions between pathogens and the cellular surface, our results suggest a parallel mechanism by which sGAGs contribute to pathogen uptake (29). In all sGAG-targeted iBMDMs we discovered a significant decrease in the surface expression of the integrins ITGB2 and ITGAL but no change in the mRNA expression. We propose that one previously overlooked function of sGAGs in pathogen uptake is maintaining the surface expression of critical receptors, including key integrins. How sGAGs regulate integrin surface expression remains unclear. Two possible models are that sGAGs stabilize interactions between surface integrins to maintain high surface expression or that sGAGs prevent rapid turnover of surface integrins. Testing these possible models will require macrophages lacking one of or combinations of sGAG-biosynthesis genes and integrins. These knockout cells will further help to delineate the epistatic relationship between sGAGs and integrins while also allowing us to investigate the specific roles of these pathways in Mab adherence. Beyond these focused studies on Mab, a broader understanding of how dynamically sGAGs control the surface proteome of cells and if this function of sGAGs contributes to other host-pathogen interactions will be of great interest.
Multiple lines of evidence from our study point toward a central role for ITGB2 in the uptake of Mab. Not only was Itgb2 a top candidate from the genome-wide screen, but we found the top enriched pathway, sGAG-biosynthesis, directly modulates ITGB2 surface expression. ITGB2 is known to complex with different integrin alpha receptors to serve as a key trafficking molecule and complement receptor (52). ITGB2, in complex with ITGAM, was previously shown to modulate LC3-associated phagocytosis (LAP) during Listeria monocytogenes infections and control the uptake of Pseudomonas aeruginosa, suggesting multiple uptake pathways intersect with ITGB2 (50,53). The identification of both Itgam and Itgb2 among the top candidates in the screen suggests a key role for this heterodimer in Mab uptake. ITGB2 also complexes with other integrins like ITGAL, yet the contribution of each of these distinct receptors in Mab uptake remains unknown and will be examined in the future. While human and murine ITGB2 proteins are highly similar, it will be important to directly test the role of this integrin in Mab uptake in human macrophages (54). In addition, investigating how Mab uptake by distinct receptors alters intracellular trafficking and/or bacterial control will better define key host-pathogen interactions during Mab infections.
How Mab mediates interactions with distinct macrophage uptake pathways remains unknown. Surface glycopeptidolipids (GPLs) are thought to contribute to macrophage infections (12,55). However, given that both smooth and rough variants of Mab required sGAG-dependent pathways for effective uptake, there must be GPL-independent mechanisms that contribute during infection. We did observe that rough variants were less affected by the loss of sGAGs than smooth variants, which may point to multiple functions of sGAGs in mediating Mab uptake. We also noted that rough variants are more susceptible to complement-mediated uptake than smooth variants. This suggests a model where GPL evade complement-mediated uptake which may modulate Mab survival. Several other Mab factors have been associated with macrophage infection, including the ATPase EccB4 and glycosyl-diacylated-nondecyl-diols (GDND), but the direct role of these genes in macrophage uptake remain unknown (15,(56)(57)(58). More broadly dissecting Mab determinants of macrophage infection will require unbiased bacterial genetic approaches, such as transposon-based approaches that were recently developed in Mab (13,15). This approach will link macrophage uptake pathways directly with specific Mabencoded factors.
In addition to understanding how Mab adheres to macrophages, the role of sGAGs during lung infection is important to consider. While Mab is mostly associated with monocytes and macrophages in vivo, current mouse models to test Mab pathogenesis remain challenging (57,59). Sustained Mab infection in mice requires multiple immune deficiencies that disrupt normal host-pathogen interactions (59). In addition, since sGAGs are essential to development, understanding how sGAGs contribute to in vivo disease will require the generation of conditional knockout animals in immune knockout backgrounds (60). While improved Mab in vivo models are being developed, the approach described here highlights a framework for leveraging unbiased genetic approaches to glean important mechanistic understanding of host-Mab interactions.
Altogether, we optimized a host genetic platform to define important macrophage pathways during Mab infection. By coupling a brightly fluorescent Mab reporter with a genome-wide knockout macrophage library, we are positioned to rapidly screen for diverse phenotypes related to macrophage-Mab interactions. Here, we leveraged this approach to dissect Mab uptake during early infection. Yet, this same approach can be used to understand host pathways that control Mab growth or contribute to macrophage cell death. Thus, our host-focused functional genetic approach will broadly identify potential host-directed therapeutic targets, with the goal of improving treatment success during Mab infection.

MATERIALS AND METHODS
Mab culture conditions, generation of fluorescent reporter, and growth assays. Isogenic pairs of smooth and rough Mab (ATTCC 19977) were used throughout this study. The Mab or E. coli mEmerald strains were built by transforming pmV261 hsp60::mEmerald into either the smooth or rough Mab variant by electroporation or DH5a E. coli by heat shock followed by selection on zeocin. All Mab cultures were grown aerobically in Middlebrook 7H9 medium supplemented with 10% Middlebrook OADC (oleic acid, dextrose, catalase, and bovine albumin) at 37°C. mEmerald GFP-expressing strains were propagated in medium containing 5 mg/mL zeocin (Invivogen).
To quantify bacterial growth, nonfluorescent and mEmerald GFP-Mab single cell suspensions were inoculated in 7H9 at starting concentration of 5x10 5 bacteria/mL. To examine antibiotic killing, rifampicin was included at a final concentration of 128 mg/mL. Optical density was quantified by measuring the absorbance at 600 nm on a Tecan Spark 20 M plate reader at the indicated time points.
Macrophage infections and uptake assays. Single cell Mab or E. coli suspensions were prepared by resuspending logarithmic phase bacteria in DMEM with 10% heat-inactivated FBS (Seradigm) followed by a soft spin (800g) to pellet large bacterial clumps. The supernatant was then used to infect macrophages, seeded at 5x10 5 /well in a 12-well plate, at the indicated MOIs for the indicated time points (2 to 6 h). Following infection, macrophages were washed with PBS, lifted from plates by scrapping, then fixed in 4% paraformaldehyde. Macrophage uptake was quantified using the BD LSRII flow cytometer (BD Biosciences) at the Michigan State University Flow Cytometry Core. Live and single macrophages were identified using forward and side scatter and the number of infected cells was determined by the fluorescence in the GFP channel. All experiments include an uninfected control to set gates for uptake quantification during analysis that was performed using FlowJo v10. For complement-mediated uptake experiments mEmerald Mab was first incubated in heat-inactivated or active FBS for 30 min prior to macrophage infections. For actin polymerization inhibitor experiments, cells were treated with DMSO or Cytochalasin D (Cayman Chemicals) for 2 h prior to infection.
For the bead uptake assay, 1 mm carboxylate-modified polystyrene latex beads expressing yellowgreen fluorescence (Sigma-Aldrich) were diluted in PBS to 5 Â 10 8 beads per mL. Beads were incubated with iBMDMs at the indicated number of latex beads per macrophage for 4 h. Following bead exposure, macrophages were washed with PBS and fixed in 4% paraformaldehyde solution. The percent uptake of yellow-green fluorescence latex beads was quantified by flow cytometry as described above.
For surface expression assays, plated cells were washed with PBS and lifted from plates by scrapping. Cells were then incubated with an Ab mixture of APC ITGAM (CD11b) and PE ITGB2 (CD18) or PE ITGB2-ITGAL (LFA1) (BioLegend; 1:400 in PBS) for 20 min at room temperature in light-free conditions. Labeled cells were then washed two times with PBS and fixed in 2% paraformaldehyde solution before relative surface expression was quantified by flow cytometry. The mean fluorescence intensity was quantified from samples using FlowJo software (BD).
CRISPR screen and analysis. Mouse Brie CRISPR knockout pooled library was a gift from David Root and John Doench (Addgene number 73633) (45) and infected with our smooth mEmerald-GFP Mab reporter strain at an MOI of five for 4 h. Following the infection, macrophages were washed with PBS then fixed with 4% paraformaldehyde. Infected library was then sorted using a Bio-Rad S3e cell sorter to isolate mEmerald1 and mEmerald-macrophages.1.5 to 2.5 Â 10 7 cells were sorted into each bin from triplicate experiments. Genomic DNA was isolated from each sorted population using Qiagen DNeasy kits after reversing DNA cross-links following a 55°C incubation overnight. Amplification of sgRNAs by PCR was performed as previously described using Illumina compatible primers from IDT (45) and amplicons were sequenced on an Illumina NextSeq 500 at the Genomics Core at Michigan State University.
Sequenced reads were first trimmed to remove any adapter sequence and to adjust for the p5 primer stagger. We used model-based analysis of genome-wide CRISPR-Cas9 knockout (MAGeCK) to map reads to the sgRNA library index without allowing for any mismatch. Subsequent sgRNA counts were median normalized to control sgRNAs in MAGeCK to account for variable sequencing depth. To test for sgRNA and gene enrichment, we used the "test" command in MAGeCK to compare the distribution of sgRNAs in the GFP 1 and GFP-bins. The MAGeCK output Gene Summary provided a ranked list of genes significantly underrepresented in the mEmerald1 bin based on 4 independent sgRNAs that was used to curate the candidate gene list.
CRISPR-targeted knockouts. Individual sgRNAs were cloned as previously described using sgOPTI that was a gift from Eric Lander (Addgene plasmid no. 85681) (45). In short, annealed oligonucleotides containing the sgRNA targeting sequence were phosphorylated, then cloned into a dephosphorylated and BsmBI (New England Biolabs) digested sgOPTI. To facilitate rapid and efficient generation of sgRNA plasmids with different selectable markers, we used sgOPTI-Blasticidin-Zeocin (BZ) and sgOPTI-Puromycin-Ampicillin (PA) as previously described (45). The simultaneous use of sgOPTI-BZ and sgOPTI-PA plasmids allowed for pooled cloning in which a given sgRNA was ligated into a mixture of BsmBI-digested plasmids. Successful transformants for each plasmid were selected by plating on ampicillin or zeocin in parallel. Next, two sgRNA-plasmid constructs per gene were packaged into lentivirus as previously described (45), then used to transduce Cas91 iBMDMs. Transductants were selected with blasticidin and/or puromycin then genomic DNA was isolated and PCR was used to amplify edited regions and sent for Sanger Sequencing (Genewiz). The resultant ABI files were used for Tracking of Idels by Decomposition (TIDE) analysis to assess the frequency and size of indels in each population compared to control macrophages (61). Three sgRNAs were targeted per gene and one targeted line was selected for follow-up studies with editing efficiency 60% to 95% for each gene. sgRNA sequences and primers for Tide analysis are included in Table S3.
Quantification of sulfated glycosaminoglycans. Sulfated Glycosaminoglycans (sGAGs) were isolated following the Blyscan Sulfated Glycosaminoglycan Assay (BioVision). In short, macrophages were washed with PBS, then 1 mL of Papain Extraction Reagent containing 1 mg/mL of Papain (Sigma-Aldrich) was added. Plates were incubated in 5% CO 2 at 37°C for 1 min. Cell Suspensions were transferred to 1.5 mL microcentrifuge tubes and immediately placed in an ice-water bath. To extract sGAGs, 200 mL of cell suspension was transferred to a 1.5 mL microcentrifuge tube and incubated at 65°C for 3 h. sGAG were then quantified using the Blyscan Sulfated Glycosaminoglycan Assay Protocol (BioVision). Briefly, 25 mL of test sample was transferred into a 1.5 mL microcentrifuge tube. Reagent blanks, sGAG standards and test samples were prepared according to the Blyscan Sulfated Glycosaminoglycan Assay General Protocol and adjusted to a 100 mL final volume. To stain sGAGs, 1 mL of Blyscan Dye Reagent (BioVision) was added to each tube, then tubes were placed in a gentle mechanical shaker for 30 min at room temperature. To pellet stained sGAGs, tubes were centrifuged at 13,000 Â g for 10 min, then supernatant was discarded. To release the recovered sGAGs, 500 mL of Dissociation Reagent (BioVision) was added to each tube and vortexed until pellets were dissolved. 200 mL of reagent blanks, sGAG standards and test samples were transferred to a 96-well plate and absorbance readings at 656 nm were obtained from an Agilent BioTek Synergy H1 Hybrid Multi-Mode Plate Reader.
RNA isolation and quantitative real-time PCR. Macrophages were resuspended in 500 mL of TRIzol reagent (Life Technologies) and incubated for 5 min at room temperature. One hundred mL of chloroform was added to the homogenate, vortexed and centrifuged at 10,000 Â g for 18 min at 4°C to separate nucleic acids. The clear, RNA containing layer was removed and combined with equal parts ethanol. This mixture was placed into a collection tube and protocols provided by the Zymo Research Direct-zol RNA extraction kit were followed. Quantity and purity of the RNA were checked using a NanoDrop and diluted to 5 ng/mL in nuclease-free water.
PCR amplification of the RNA was completed using the One-step Syber Green RT-PCR kit (Qiagen). 25 ng of total RNA was added to a master mix reaction of the provided RT mix, Syber green, gene specific primers (5 mM of forward and reverse primer), and nuclease-free water. For each biological replicate (triplicate), reactions were conducted in technical triplicates in 96-well plates. PCR product was monitored using the QuantStudio3 (ThermoFisher). The number of cycles needed to reach the threshold of detection (Ct) was determined for all reactions. The mean CT of each experimental sample in triplicate was determined. The average mean of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was subtracted from the experimental sample mean CT for each gene of interest (dCT).
Bioinformatics analysis. Gene set enrichment analysis (GSEA) was used to identify enriched pathways in the MAGeCK analyzed data set based on the full ranked list as done previously. For GSEA analysis, the "GSEA Preranked" function was used to complete functional enrichment using default settings for Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology terms. DAVID analysis was used to identify enriched pathways within the positive regulators of Mab uptake. Using the robust rank aggregation (RRA) the top enriched positive regulators (2-fold change with at least 3 sgRNAs) were used as a "candidate list." Functional analysis and functional annotation analysis were completed, and top enriched pathways and protein families were identified.
Statistical analysis. Statistical analysis and data visualization were performed using Prism version 9 (GraphPad Software) or Biorender, as indicated in figure legends. Data are presented, unless otherwise indicated, as the mean 6 SD and individual data points are shown for each experiment. For parametric data, one-way or two-way ANOVA followed by Tukey's or Dunnett's multiple-comparison test were used to identify significant differences between multiple groups.
Data availability. Raw sequencing data in FASTQ and processed formats are available for download from NCBI Gene Expression Omnibus (GEO) accession number GSE221413.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.