Pharmacological inhibition of host pathways enhances macrophage killing of intracellular bacterial pathogens

Pharmacological inhibition of host pathways enhances macrophage killing of intracellular bacterial pathogens | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Pharmacological inhibition of host pathways enhances macrophage killing of intracellular bacterial pathogens Ramesh Rijal , View ORCID Profile Richard H. Gomer doi: https://doi.org/10.1101/2025.04.06.647500 Ramesh Rijal 1 School of Biological, Environmental, and Earth Sciences, The University of Southern Mississippi , Hattiesburg, Mississippi, USA 2 Department of Biology, Texas A&M University, College Station , Texas, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: Ramesh.Rijal{at}usm.edu Richard H. Gomer 2 Department of Biology, Texas A&M University, College Station , Texas, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Richard H. Gomer Abstract Full Text Info/History Metrics Preview PDF Abstract After ingestion into macrophage phagosomes, some bacterial pathogens such as Mycobacterium tuberculosis ( Mtb ) evade killing by preventing phagosome acidification and fusion of the phagosome with a lysosome. Mtb accumulates extracellular polyphosphate (polyP), and polyP inhibits macrophage phagosome acidification and bacterial killing. In Dictyostelium discoideum , polyP also inhibits bacterial killing, and we identified some proteins in D. discoideum that polyP requires to suppress the killing of ingested bacteria. Here, we find that pharmacological inhibition of human orthologues of the D. discoideum proteins, including P2Y1 receptors, mammalian Target of Rapamycin (mTOR), and inositol hexakisphosphate kinase, enhances the killing of Mtb , Legionella pneumophila , and Listeria monocytogenes by human macrophages. Mtb inhibits phagosome acidification, expression of the proinflammatory marker CD54, and autophagy, and increases expression of the anti-inflammatory marker CD206. In Mtb -infected macrophages, the polyP-degrading enzyme polyphosphatase (ScPPX) and inhibitors reversed these effects, with ScPPX increasing CD54 expression more in female macrophages compared to male macrophages. In addition, Mtb inhibits proteasome activity, and some, but not all, inhibitors reversed these effects. While the existence of a dedicated polyP signaling pathway remains uncertain, our findings suggest that pharmacological inhibition of select host proteins can restore macrophage function and enhances the killing of intracellular pathogens. Importance Human macrophages engulf bacteria into phagosomes, which then fuse with lysosomes to kill the bacteria. However, after engulfment, pathogenic bacteria such as Mycobacterium tuberculosis , Legionella pneumophila , and Listeria monocytogenes can block phagosome-lysosome fusion, allowing their survival. Here, we show that pharmacological inhibition of specific macrophage proteins reverses these effects and enhances bacterial killing. These findings suggest that targeting host factors involved in these processes may provide a therapeutic strategy to improve macrophage function against infections such as tuberculosis, Legionnaires’ disease, and listeriosis. Introduction Macrophages engulf invading microorganisms into a membrane-bound compartment called the phagosome ( 1 – 3 ). The phagosome then acidifies and fuses with lysosomes to form a phagolysosome ( 4 ). Within the phagolysosome, hydrolytic enzymes, antimicrobial compounds, and reactive oxygen and nitrogen species kill the ingested pathogen ( 4 , 5 ). Pathogens such as Mycobacterium tuberculosis ( Mtb ) can prevent the fusion of phagosomes with lysosomes in macrophages, thereby evading the bactericidal actions of the lysosome and enabling them to persist within macrophages ( 6 , 7 ). Listeria monocytogenes (referred to as Listeria hereafter), the bacterium that causes listeriosis, escapes from the phagosome into the cytosol, thus avoiding killing in the phagolysosome ( 8 ). Legionella pneumophila (referred to as Legionella hereafter), the bacterium that causes Legionnaires’ disease, employs secreted factors to manipulate host cell processes and evade immune detection, allowing it to establish replicative vacuoles within host cells ( 9 ). Determining how these bacteria prevent macrophages from killing them could reveal strategies to enhance macrophage bactericidal function. Polyphosphate (polyP) is a linear chain of phosphate units found across all forms of life, from bacteria to humans, playing crucial roles in energy storage, stress response, and metabolism ( 10 ). Polyphosphate kinase (PPK), a widely conserved bacterial enzyme, catalyzes the synthesis of polyP from ATP ( 11 ). PolyP levels are also affected by polyphosphatase (PPX), an enzyme that degrades polyP ( 12 ). Pathogenic bacteria lacking PPK, or having reduced PPK levels, exhibit defects in stress response, quorum sensing, growth, survival, and virulence ( 13 – 20 ). Mtb possesses PPK1 and PPK2 enzymes, which are absent in humans ( 21 ). Intracellular polyP is necessary for the survival of Mtb in host cells ( 18 , 22 – 24 ), and deletion of PPK1 in M. smegmatis (Msmeg) attenuates the survival of ingested Msmeg in human macrophages ( 25 ). In addition to intracellular polyP, Mtb and Neisseria gonorrhoeae, the bacteria that causes gonorrhea, have polyP in their envelopes, and this envelope-associated polyP appears to protect these bacteria from antimicrobials ( 19 , 20 , 26 ). We previously found that Mtb also accumulate extracellular polyP, and calculations suggested that the concentration of polyP in the space inside a phagosome and outside the Mtb cell would quickly rise ( 25 ). Treatment of Mtb -infected macrophages with recombinant polyphosphatase (ScPPX) reduced the Mtb burden in macrophages ( 25 ), suggesting that both intracellular and extracellular polyP potentiate the survival of Mtb in host cells. Bacterial polyP also impairs macrophage function by interfering with proinflammatory polarization and suppressing type I interferon responses, thereby promoting bacterial survival ( 27 ). Treatment of Mtb with gallein, a broad-spectrum PPK inhibitor ( 28 – 30 ), potentiates the ability of isoniazid, an anti- Mtb antibiotic, to inhibit Mtb growth in both in vitro culture and within human macrophages ( 26 ). Together, these findings suggest that bacterial polyP may modulate macrophage responses in ways that enhance bacterial survival. PolyP inhibits phagosome acidification and lysosome activity in the eukaryotic microbe Dictyostelium discoideum and human macrophages ( 25 ). Exogenous polyP promotes the survival of bacteria in D. discoideum , and by examining this effect in known mutants, we found that polyP requires the mTOR complex protein Lst8, the inositol hexakisphosphate kinase I6kA, and the Rho-GTPase RacE to facilitate survival of ingested bacteria ( 25 , 31 ). Based in part on the polyP pathway identified in D. discoideum , we tested pharmacological inhibitors targeting the human orthologs of these proteins and found that several of these compounds potentiate the ability of macrophages to kill Mtb , Listeria , and Legionella . Results MRS2279 and TNP potentiate the ability of macrophages to kill Mtb, Legionella, and Listeria In D. discoideum , a signal transduction pathway appears to mediate the ability of polyP to enhance the survival of ingested bacteria ( 31 ). To determine whether similar mechanisms operate in macrophages, we tested pharmacological inhibitors targeting both human homologs of Dictyostelium proteins required for polyP-mediated bacterial survival and other macrophage proteins reported to interact with polyP. Table 1 lists proteins that may mediate polyP sensing. In humans, the purinergic receptor P2Y1 senses polyP ( 32 ), and the drug MRS2279 inhibits this receptor ( 33 , 34 ). The human Receptor for Advanced Glycation (RAGE) also senses polyP ( 32 ), and FPSZM1 inhibits RAGE ( 35 ). For Lst8, IP6K, RacE, and RhoA (proteins identified in the D. discoideum screen) there are human orthologues for which inhibitors have been found. Rapamycin inhibits human mTORC1 ( 36 ), TNP inhibits human IP6K ( 37 ), MBQ167 inhibits the small GTPases Rac/Cdc42 ( 38 ), Rhosin and Y16 inhibit the small GTPase RhoA ( 39 , 40 ). AdcB (UniProt: Q86KB1; another protein identified in the D. discoideum screen) does not have a clear human ortholog, but Alphafold/ Foldseek analysis suggested that human thioredoxin-interacting protein is a structural homolog of Dictyostelium AdcB ( 41 – 43 ). Extendin-4 inhibits human thioredoxin-interacting protein ( 44 , 45 ). View this table: View inline View popup Download powerpoint Table 1: Inhibitors for human proteins or human orthologs of D. discoideum proteins identified in ( 31 ). Granulocyte macrophage-colony stimulating factor (GM-CSF) causes monocytes to become pro-inflammatory macrophages, whereas macrophage colony-stimulating factor (M-CSF) causes monocytes to become anti-inflammatory macrophages ( 46 ). Human blood monocytes were cultured for 6 days with GM-CSF or M-CSF to generate macrophages, and these were allowed to phagocytose Mtb in the presence of ScPPX or the drugs listed in Table 1 , washed, treated with 200 µg/ ml gentamicin (Sigma, St. Louis, MO) to kill uningested bacteria, and then lysed at 4 hours or at 48 hours with a detergent that does not kill bacteria. The ScPPX or drugs were present in all the washes and throughout the experiment. The lysates were plated to measure the number of viable bacteria. We previously observed that ScPPX reduces the ingested Mtb burden in GM-CSF macrophages at 48 hours ( 25 ). In GM-CSF macrophages at 4 hours (relatively shortly after ingesting bacteria), 1000 nM MRS2279 had no significant effect on the number of ingested Mtb that were still viable, but 1000 nM TNP increased the number of Mtb ( Figure 1A ). At 48 hours, 1000 nM MRS2279 or 1000 nM TNP reduced the number of viable ingested Mtb ( Figure 1B ). At 4 hours, 10 µg/ml ScPPX, 1000 nM FPSZM1, or 1000 nM Rapamycin did not significantly alter the number of viable ingested Mtb ( Figure S1A ), whereas at 48 hours all three reduced the number of viable ingested Mtb ( Figure S1B ). At 48 hours, 1000 nM MBQ167, 1000 nM Rhosin, 1000 nM Y16, or 1000 nM Extendin-4 did not significantly affect the viability of ingested Mtb ( Figure S1C ). Download figure Open in new tab Figure S1: ScPPX, Rapamycin, or FPSZM1 potentiates the ability of macrophages to kill ingested Mtb. Viable ingested Mtb in macrophages with GM-CSF, in the absence (Control) or presence of 10 µg/ml ScPPX or 1000 nM of the indicated inhibitor, was determined as colony-forming units (CFU) at 4 hours (A) or 48 hours (B and C) after ingestion. For each experiment with each donor, CFU in the control was considered 100%. All values are mean ± SEM of six (three female and three male donors) independent experiments. Male data points are shown in blue and female data points are shown in red. For each bar, there was no significant difference between male and female (unpaired t-test). * p < 0.05 compared to control (one-way ANOVA, Dunnett’s test). Download figure Open in new tab Figure 1: MRS2279 or TNP potentiates the ability of macrophages to kill ingested Mtb , Legionella , or Listeria . (A – F) Viable ingested bacteria in macrophages with GM-CSF or M-CSF, in the absence (Control) or presence of the indicated concentration of MRS2279 and TNP, was determined as colony-forming units (CFU) at 4 hours (A), 48 hours (B, E, and F), or 72 hours (C and D) after ingestion. For each experiment with each donor, CFU in the control was considered 100%. All values are mean ± SEM of six (three female and three male) (A – D and F) or three (one female and two male) (E) independent experiments. Male data points are shown in blue and female data points are shown in red. For each bar, there was no significant difference between male and female (unpaired t-test). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 compared to the 0 control (one-way ANOVA with Dunnett’s test for A and B, and two-way ANOVA with Dunnett’s test for C – F). To determine if longer treatment times also decrease the viability of Mtb , we lysed cells at 72 hours. Compared to control, 100 or 1000 nM MRS2279, and 10, 100, or 1000 nM of TNP reduced the number of viable ingested Mtb in GM-CSF macrophages ( Figure 1C ). All concentrations of MRS2279, or TNP also reduced the number of viable ingested Mtb at 72 hours in M-CSF macrophages ( Figure 1D ). To determine if the drugs have similar effects on ingested Legionella and Listeria , GM-CSF macrophages were allowed to phagocytose Legionella or Listeria in the absence or presence of MRS2279 or TNP, and the viability of ingested bacteria was tested at 48 hours. 100 or 1000 nM MRS2279 and 10, 100, or 1000 nM TNP reduced the number of viable ingested Legionella ( Figure 1E ). Similarly, 10 or 1000 nM MRS2279, and 10, 100, or 1000 nM TNP reduced the number of viable ingested Listeria ( Figure 1F ). For all the above results, there were no significant differences between macrophages from male donors (blue symbols in graphs) and female donors (red symbols). Together, these data suggest that pharmacological inhibition of multiple macrophage proteins, identified based on a putative polyP-responsive pathway, potentiate the killing of ingested bacteria, supporting the idea that targeting host pathways can restore macrophage killing of intracellular pathogens. MRS2279 and TNP do not affect the viability of macrophages or uningested Mtb, Legionella, or Listeria The viability of human macrophages can be tested by incubating cells with Deep Blue Cell Viability resazurin dye ( 47 ). Metabolically active cells reduce blue resazurin to the pink product resorufin ( 47 ). With this assay, none of the inhibitors significantly altered the metabolic activity of uninfected or infected macrophages ( Figures S2A-C ). We also monitored the optical density, as a measure of growth, of Mtb , Legionella , or Listeria in the presence of 1000 nM of the inhibitors with no macrophages present. None of the inhibitors tested significantly affected bacterial growth ( Figures S2 D-F ). Together, these results suggest that the inhibitors reduce the viability of ingested bacterial pathogens by enhancing macrophages’ ability to kill the ingested bacteria and not by inhibiting macrophage viability or bacterial growth. Download figure Open in new tab Figure S2: ScPPX or inhibitors do not significantly affect metabolic activity of infected macrophages or growth of bacteria. (A – C) Uninfected macrophages or Mtb , Legionella , or Listeria infected macrophages in the absence or presence of 10 µg/ml ScPPX or the indicated inhibitor for 24 hours were incubated with Deep Blue Cell Viability resazurin dye for 12 hours, and fluorescence was measured. For each experiment with each donor, the average of the metabolic activity of uninfected macrophages was considered 100%. (D – F) Mtb , Legionella , or Listeria cultures were grown for 6 days ( Mtb ), 144 hours (Legionella), or 48 hours ( Listeria ) in the absence (Control) or presence of 1000 nM of the indicated inhibitor. The OD 600 was measured daily. Values are mean ± SEM of six (three females and three males) (A and B), five (two female and three male) (C), or mean ± SD of six (D – F) independent experiments. For A – C, male data points are shown in blue and female data points are shown in red. For each bar in A-C, there was no significant difference between male and female (unpaired t-test). MRS2279 and TNP block effects of exogenous polyP Exogenous polyP inhibits the ability of Dictyostelium and human macrophages to kill ingested E. coli , which do not accumulate detectable levels of extracellular polyP ( 25 ). To determine whether this effect of exogenous polyP can be reversed, GM-CSF macrophages were allowed to phagocytose E. coli in the absence or presence of 15 µg/ml exogenous polyP, with or without selected inhibitors. At 4 hours, polyP in the absence (no drug) or presence of MRS2279, FPSZM1, rapamycin, or TNP did not significantly affect the number of viable ingested E. coli ( Figure 2A ). At 48 hours, polyP with no drug increased the number of viable ingested E. coli , and MRS2279, FPSZM1, rapamycin, and TNP partially prevented this effect ( Figure 2B ). Possibly because ScPPX and polyP increased the number of ingested E. coli at 4 hours ( Figure 2A ), ScPPX in the presence of exogenous polyP did not significantly decrease the viability of ingested E. coli at 48 hours ( Figure 2B ). As above, there was no significant effect of blood donor sex on the results. These data suggest that the enhanced survival of ingested E. coli caused by exogenous polyP can be partially reversed by pharmacological inhibitors targeting host proteins that may mediate macrophage responses to polyP. Download figure Open in new tab Figure 2: Inhibitors prevent polyP-induced survival of ingested E. coli and reduced phagosome acidification. (A – B) Viable E. coli in GM-CSF macrophages, in the absence (Control) or presence of 15 µg/ml polyP, without (No drug) or with 1000 nM of the indicated inhibitor, was determined as colony-forming units (CFU) at 4 hours (A) or 48 hours (B). CFU in all treatment conditions were compared with the No drug condition for statistical significance. (C) A schematic showing macrophages with phagosomes containing pHrodo red-labeled yeast, in the presence or absence of polyP, with or without MRS2279 or TNP. pHrodo red-labeled yeast have low fluorescence outside the cell but show red fluorescence in acidic phagosomes. (D) Human GM-CSF macrophages were incubated with yeast, in the absence (Control) or presence of 15 µg/ml polyP without (No drug) or with 1000 nM of the indicated inhibitor (+ polyP) for 1 hour, fixed, and fluorescence images were taken. 100 nM of Concanamycin A (ConcanA) is a positive control for inhibition of phagosome acidification. Differential interference contrast (DIC) merged with fluorescence are at the left, and fluorescence images are at the right for each treatment condition. Bar is 20 µm. Images are representative of six independent experiments (three females and three males). (E – G) Images from D were used to measure fluorescence intensities of yeast (E), percent of macrophages with yeasts (F), and number of yeasts per macrophages (G). The fluorescence intensity of yeast in control was set to 100 for E. (H – K) Experiments from D – G were performed with M-CSF macrophages. Size bars are 20 µm. Male data points are shown in blue and female data points are shown in red. For each bar, there was no significant difference between male and female (unpaired t-test). All values are mean ± SEM of six (three females and three males) independent experiments. * or # p < 0.05; ** p < 0.01; ### p < 0.001; #### p < 0.0001 (one-way ANOVA with Dunnett’s test). * indicates compared to no drug; # indicates compared to control. MRS2279 and FPSZM1 prevent polyP mediated inhibition of phagosome acidification Bacterial pathogens, including Mtb , have evolved ways to prevent phagosome acidification ( 4 , 7 , 48 ), and exogenous polyP also inhibits phagosome acidification in human macrophages ( 25 ). To determine whether inhibitors could reverse this effect, GM-CSF or M-CSF macrophages were incubated with pHrodo red-labeled dead yeast particles. The pHrodo red-labeled particles (referred to as yeast hereafter) is non-fluorescent outside the cell but fluoresces brightly red in acidic phagosomes ( 49 ) ( Figures 2C and S3A ). Concanamycin A (concanA), a vacuolar-type H+-ATPase inhibitor that blocks phagosome acidification ( 50 ), served as a positive control for inhibiting phagosome acidification ( Figures 2D-K ). As previously observed ( 25 ), polyP reduced the fluorescence of ingested yeast in macrophages ( Figures 2D, E, H, I ), and concanA also reduced the fluorescence of ingested yeast in macrophages ( Figures 2D, E, H, I ). PolyP and concanA did not significantly affect the percentage of macrophages with yeasts or the number of ingested yeasts per macrophage ( Figures 2F, G, J , and K ). In the presence of polyP, 1000 nM MRS2279 increased the fluorescence intensities of ingested yeast in GM-CSF and M-CSF macrophages ( Figures 2D, E, H , and I ), and 1000 nM FPSZM1 increased the fluorescence intensities of ingested yeast in M-CSF macrophages ( Figures S3F and G ). Neither polyP nor the inhibitors significantly altered the percentage of macrophages with ingested yeast ( Figures 2F , 2J , S3D, and S3H) or the number of ingested yeasts per macrophage ( Figures 2G , 2K , S3E, and S3I). Rapamycin or FPSZM1 did not significantly reverse the effect of polyP on the number of acidic phagosomes per cell in GM-CSF macrophages ( Figures S3B and C ). There was no significant effect of blood donor sex on the results. Together, these data suggest that inhibitors such as MRS2279 and FPSZM1 can prevent polyP’s effect on phagosome acidification. Download figure Open in new tab Figure S3: FPSZM1 prevents polyP-mediated inhibition of phagosome acidification in M-CSF macrophages. (A) Human macrophages (Mφ) with GM-CSF were incubated without (top) or with yeast (bottom) for 1 hour, fixed, and fluorescence images were taken as in Figures 2 D and H . Differential interference contrast (DIC) images are at the left, DIC merged with fluorescence are in the middle, and fluorescence images of macrophages without (top) and with yeasts (bottom) are at the right. Bar is 20 µm. Images are representative of five independent experiments (two female and three male). (B - I) Human macrophages with GM-CSF (B – E) or M-CSF (F – I) were incubated with yeast, in the absence (Control) or presence of 15 µg/ml polyP (+ polyP) without (No drug) or with 1000 nM of the indicated inhibitor for 1 hour, fixed, fluorescence images were taken, and fluorescence intensities of yeast, percent of macrophages with yeasts, and number of yeasts per macrophages were determined as in Figure 2 D – K . DIC merged with fluorescence are at the left, and fluorescence images are at the right for each treatment condition. Bars are 20 µm. Images are representative of five independent experiments (two female and three male). Male data points are shown in blue and female data points are shown in red. For each bar, there was no significant difference between male and female (unpaired t-test). All values are mean ± SEM of five (two female and three male) independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA with Dunnett’s test). Inhibition of polyP-related host responses enhances proinflammatory polarization Enhanced expression of CD54, also known as intercellular adhesion molecule 1 (ICAM-1), is associated with the polarization of macrophages toward a proinflammatory phenotype with enhanced phagocytosis ( 51 , 52 ). CD54 expression is decreased in Mtb -infected host cells ( 53 ). CD206, also known as the mannose receptor, is a cell surface pattern recognition receptor primarily expressed in anti-inflammatory macrophages and is involved in detecting and phagocytosing pathogens such as Mtb ( 54 ). Compared to the control, both polyP or Mtb alone reduced anti-CD54 staining and increased anti-CD206 staining of GM-CSF macrophages ( Figures 3A-D ). The presence of 10 µg/ml ScPPX, or 1000 nM MRS2279, FPSZM1, Rapamycin, or TNP in macrophage- Mtb coculture increased CD54 staining and decreased CD206 staining on macrophages ( Figures 3A-D ), with ScPPX showing more of an effect on increasing CD54 expression in female macrophages compared to male macrophages. Together, these data suggest that except for ScPPX, all the inhibitors reduce the ability of Mtb to inhibit macrophage polarization away from a proinflammatory phenotype and reduce the ability of Mtb to increase CD206 expression in a sex-independent manner. Download figure Open in new tab Figure 3: Some inhibitors enhance the proinflammatory polarization of macrophages. Uninfected (control), 15 µg/ml polyP treated, or Mtb infected macrophages with GM-CSF in the absence (No drug) or presence of 10 µg/ml ScPPX or 1000 nM of the indicated inhibitor at 24 hours after phagocytosis (as described in the bacterial survival assay) were fixed, permeabilized and stained with antibodies against CD54 or CD206, and fluorescence intensities were measured and average of control (B) or No drug (D) was considered 100%. Bars are 100 µm. Fluorescence images are representative of six independent experiments (three female and three male). All values are mean ± SEM of six (three females and three males) independent experiments. One or more than one images from each independent experiment were analyzed, indicated by multiple data points. Male data points are shown in blue and female data points are shown in red. An unpaired t-test was performed to assess male vs. female differences for each bar in the graph. * p < 0.05; ** or ## p < 0.01; **** p < 0.0001 (Kruskal-Wallis test). *indicates compared to no drug; # indicates compared to control. $ p < 0.05 indicates female vs male (unpaired t-test). Inhibitors restore proteasome activity suppressed by polyP or Mtb The proteasome generates peptides for antigen presentation by MHC Class I molecules ( 55 , 56 ). Mtb inhibits antigen presentation and the induction of adaptive immune responses ( 57 ). We previously found that polyP inhibits proteasome activity in Dictyostelium , and this requires the mTOR protein Lst8, RacE, I6kA, the arrestin protein AdcB, and the polyP receptor GrlD ( 31 ). To determine if inhibitors affect proteasome activity, we measured proteasome activity in macrophages treated with polyP or infected with Mtb in the presence or absence of the inhibitors described above. Female macrophages showed increased basal proteasome activity compared to male macrophages ( Figure 4A ). The proteasome inhibitor Mg132 ( 55 ) inhibited proteasome activity, and both polyP and Mtb reduced proteasome activity ( Figure 4B ). The presence of 1000 nM MRS2279 partially restored proteasome activity in Mtb -infected macrophages, whereas 10 µg/ml ScPPX, 1000 nM FPSZM1, 1000 nM TNP, or 1000 nM rapamycin did not significantly alter proteasome activity ( Figure 4B ). Together, these data suggest that Mtb may suppress macrophage proteasome activity via a mechanism involving polyP, and that some host-targeted inhibitors can counteract this effect. Download figure Open in new tab Figure 4: Some inhibitors counteract Mtb inhibition of proteasome activity and potentiate autophagy. (A and B) Proteasome activity of uninfected (control), 4 nM Mg132 or 15 µg/ml polyP treated, or Mtb infected macrophages in the absence (No drug) or presence of 1000 nM of the indicated inhibitor were assessed at 24 hours after phagocytosis. The proteasome inhibitor Mg132 was used as a control. (C) The lysates of macrophages from B were also resolved by SDS/PAGE, and Western blots of lysates were stained for the indicated protein. Representative blots from six independent experiments are shown. Although TNP blots were part of the same membrane, they are presented separately for clarity. GAPDH staining of samples indicates loading controls. (D) Band intensities from C were quantified by densitometric analysis. The intensity of the control was set to 100. All values are mean ± SEM of six (three females and three males) independent experiments. Male data points are shown in blue and female data points are shown in red. * or # p < 0.05; ** p < 0.01; *** or ### p < 0.001 (one-way ANOVA with Dunnett’s test (B and D). *indicates compared to no drug (B) or control (D); # indicates compared to control (A); $ indicates p < 0.01 female compared to male (unpaired t-test). Some inhibitors enhance LC3-II accumulation in Mtb-infected macrophages In addition to affecting phagosome acidification and lysosome activity, Mtb induces phagosome permeabilization ( 58 ). Macrophages recognize damaged phagosomes and attempt to route these damaged phagosomes to the autophagosome through selective autophagy ( 59 ). Autophagy is mediated by the recruitment of autophagy machinery components and the autophagy-activating microtubule-associated light chain 3 (LC3) ( 60 ). LC3 undergoes conjugation with phosphatidylethanolamine, resulting in the formation of LC3-II, which is found in autophagosomes ( 61 ). LC3-II is a marker for autophagosomes since the quantity of LC3-II directly correlates with the count of autophagosomes and structures related to autophagy ( 62 ). However, Mtb blocks autophagy by inhibiting the recruitment of autophagy machinery components and the autophagy-activating microtubule-associated light chain 3 (LC3) ( 60 ). In agreement with the idea that Mtb blocks autophagy, we observed that Mtb did not significantly alter the level of LC3-II ( Figures 4C and D ). PolyP also did not significantly affect levels of LC3-II, but MRS2279, TNP, or Rapamycin increased the level of LC3-II in macrophages with ingested Mtb , indicating increased autophagy ( Figures 4C and D ). ScPPX did not significantly alter the level of LC3-II. Together, these data suggest that Mtb may suppress autophagy, and that selected inhibitors can counteract this effect. Discussion In this report, we have shown that pharmacological inhibitors targeting the human polyP receptors P2Y1 or RAGE, and mTOR and I6KA, human orthologs of Dictyostelium proteins required for polyP-mediated bacterial survival, attenuate the survival of ingested Mtb in human macrophages, and inhibitors of P2Y1 and I6KA attenuate the survival of ingested Legionella or Listeria. These results suggest that some host responses to bacterial polyP may be conserved between D. discoideum and human macrophages, and that targeting these host components could offer a therapeutic strategy to enhance macrophage-mediated clearance of intracellular pathogens. Listeria secretes listeriolysin, which forms pores in the phagosome, and this allows the bacteria to move from the phagosome to the cytosol shortly after infection ( 63 ). Possibly because of this, the viability of Listeria appeared to be relatively less affected compared to Mtb and Legionella in the presence of MRS2279 or TNP. CD54 on macrophages enhances their phagocytic capabilities, particularly under inflammatory conditions induced by stimuli such as lipopolysaccharide (LPS). This enhancement is linked to the production of reactive oxygen species mediated by Toll-like receptor 4 (TLR4) signaling ( 52 ). The reduced expression of CD54 by Mtb and polyP suggests a mechanism by which these pathogens modulate macrophage polarization. However, the presence of ScPPX or inhibitors such as MRS2279, FPSZM1, TNP, and rapamycin restored CD54 expression, indicating that inhibiting macrophage components can counteract the immune evasion strategies of Mtb by promoting a proinflammatory macrophage phenotype. CD206, also known as the macrophage mannose receptor, is involved in recognizing and binding to carbohydrate structures on the surface of Mtb ( 64 – 66 ). Once Mtb binds to CD206, this interaction limits phagosome-lysosome fusion, helping the bacteria to survive within macrophages ( 66 ). Both Mtb and polyP increased CD206 expression, but this effect was reduced by ScPPX, MRS2279, FPSZM1, TNP, and rapamycin. However, among these, only MRS2279 and FPSZM1 reversed the inhibitory effect of polyP on phagosome acidification, suggesting that polyP may suppress phagosome acidification through a mechanism that does not require elevated CD206 levels or operates through partially distinct host responses. The proteasome is crucial for the degradation of intracellular proteins into peptide fragments suitable for presentation by MHC Class I molecules, which are essential for the activation of cytotoxic T lymphocytes and the adaptive immune response ( 67 , 68 ). Our findings suggest that Mtb may suppress proteasome activity in macrophages, potentially through mechanisms involving polyP. MRS2279 increased proteasome activity in Mtb -infected macrophages, whereas ScPPX, FPSZM1, TNP, and rapamycin did not. PolyP may trigger downstream pathways, such as purinergic signaling via P2Y1 receptors, that continue even after partial polyP degradation. Therefore, ScPPX’s action may be too late to reverse signaling that has already been initiated by polyP. The lack of effect from TNP suggests that IP6K inhibition alone may be insufficient, possibly due to compensatory pathways such as those involving mTOR or Rho GTPases. Rapamycin may suppress proteasome function by inhibiting mTOR activity, which is known to regulate proteasome biogenesis ( 36 ). RAGE inhibition by FPSZM1 could also affect proteasome regulation indirectly, given its broader role in modulating inflammatory signaling ( 69 , 70 ). These findings indicate that polyP may impair proteasome activity through multiple host pathways or Mtb may inhibit proteasome activity through polyP-independent mechanisms, and that selective targeting of specific host factors may help restore proteasome function in Mtb -infected macrophages. Autophagy is a critical host defense mechanism that targets intracellular pathogens such as Mtb for degradation ( 71 , 72 ). LC3-II, the lipidated form of LC3 that associates with autophagic membranes, is involved in autophagosome maturation and cargo selection, the later stages of autophagy ( 73 ). Rapamycin promotes autophagy by relieving mTOR-mediated suppression of autophagy initiation ( 74 ). Similar to the treatment with rapamycin, MRS2279 and TNP increased LC3-II levels, suggesting enhanced autophagic activity. This increase in LC3-II levels indicates that these inhibitors can overcome Mtb ’s blockade of autophagy induction. In contrast, ScPPX did not significantly affect LC3-II levels, indicating that its effect on bacterial clearance may involve autophagy-independent mechanisms. In conclusion, our findings suggest that targeting specific host proteins influenced by bacterial polyP can restore key macrophage functions, such as phagosome acidification, proteasome activity, and autophagy. This host-directed approach may enhance the clearance of intracellular pathogens including Mtb , Legionella , and Listeria . Future studies will be needed to determine how polyP levels and the effects of these inhibitors vary across different bacterial species and clinical isolates. Materials and Methods Inhibitors 10 mM stocks of MRS2279 (Cat#2158, Tocris, Minneapolis, MN) ( 33 , 34 ), TNP (Cat#3946 Tocris) ( 37 ), FPS-ZM1 (Cat# 6237, Tocris) ( 35 ), Rapamycin (Cat# J62473, Alfa Aesar, Ward Hill, MA) ( 36 ), MBQ167 (Cat#HY-112842, MedChemExpress, Monmouth Junction, NJ) ( 38 ), Rhosin (Cat#5003, Tocris) ( 39 ), Y16 (Cat#SML0873, Sigma, St. Louis, MO) ( 40 ), and Exendin-4 (Cat#Enz-PRT111-0500, Enzo Life Sciences, Farmingdale, NY) ( 44 , 45 ) were prepared in water or DMSO according to the manufacturer’s instructions. Aliquots of 50 µl were stored at -20°C and were diluted in the macrophage culture media described below. Human cell culture Human peripheral blood was collected from healthy volunteers who gave written consent, and with specific approval from the Texas A&M University human subjects institutional review board. Peripheral blood mononuclear cells (PBMCs) were purified as previously described ( 75 ). The PBMCs were cultured in RBCSG (Roswell Park Memorial Institute (RPMI) medium (12-167F, Lonza, Walkersville, MD) containing 10% bovine calf serum (2100-500, VWR Life Science Seradigm, Radnor, PA) and 2 mM l-glutamine (Lonza)), and where indicated containing 25 ng/mL human granulocyte-macrophage colony-stimulating factor (572903 GM-CSF) or 25 ng/mL human macrophage colony-stimulating factor (574804 M-CSF) (Biolegend, San Diego, CA) at 37 °C in a humidified chamber with 5% CO 2 in type 353219, 96-well, black/clear, tissue-culture-treated, glass-bottom plates (Corning, Big Flats, NY) with 10 5 cells per well in 100 µL or type 353072, 96-well, tissue-culture-treated, polystyrene plates (Corning) with 10 5 cells per well in 100 µL. At day 7, loosely adhered cells were removed by gentle pipetting, and fresh RBCSG containing GM-CSF or M-CSF was added to the cells to a final volume of 100 µL per well. Bacterial cell culture The Biosafety Level-1 strain of E. coli K-12 (BW25113) (CGSC#7636) ( 76 , 77 ), from the E. coli genetic stock center ( 76 , 77 ), was grown at 37 °C in Luria-Bertani (LB) broth (BD, Sparks, MD) in a 50 ml conical tube (VWR) or on LB Agar on a type 25384-302 petri dish (VWR) at 37 °C in a humidified incubator for 1 day. The attenuated (mc-ΔleuDΔpanCD) Biosafety Level-2 strain of Mtb (a derivative of the H37Rv strain) ( 78 ) (a gift from Dr. Jim Sacchettini, Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX) was grown as described ( 79 ) in Middlebrook 7H9 broth (BD, Sparks, MD) in a type 89039-656 50 ml conical tube (Falcon, VWR Life Science Seradigm) or on 7H10 agar (BD) at 37 °C in a humidified incubator. Both Mtb media contained 0.5% glycerol (VWR), 0.05% Tween 80 (MP Biomedicals, Solon, OH), and the Middlebrook Oleic ADC Enrichment (BD). Mtb ΔleuDΔpanCD cultures (both liquid and agar plates) were additionally supplemented with 50 μg/mL leucine (VWR Life Science Seradigm) and 50 μg/mL pantothenate (Beantown Chemical, Hudson, NH). Liquid cultures were incubated in 50 ml conical tubes on a STR200-V variable angle tube rotator (Southwest Science, Roebling, NJ) for 1 to 2 weeks until the cell density reached log phase, and the agar plates were wrapped in plastic film to prevent desiccation and incubated for 3 to 4 weeks at 37°C in a humidified incubator. The Biosafety Level-2 strain of Legionella ( Legionella pneumophila subsp. pneumophila Brenner et al.) (American Type Culture Collection (ATCC) 33153) was grown as described by ATCC ( https://www.atcc.org/products/33153 ) in liquid 1099 CYE Buffered Medium or a solid 1099 CYE Buffered Medium at 37 °C in a humidified incubator with 5% CO 2. for 3 days. The Biosafety Level-2 strain of Listeria ( Listeria monocytogenes (Murray et al.) Pirie))(ATCC 19111) was grown as described by ATCC ( https://www.atcc.org/products/19111 ) in a 44 Brain Heart Infusion Broth or on 44 Brain Heart Infusion Agar at 37 °C in a humidified incubator for 2 days. Recombinant polyphosphatase and polyP The plasmid for purifying S. cerevisiae exopolyphosphatase (ScPPX) was a kind gift from Michael Gray, University of Alabama at Birmingham, AL ( 80 ). Recombinant ScPPX was purified as previously described in a protein purification protocol ( 81 ). 10 µg/ ml of ScPPX was used to treat human macrophages in the assays. Long chain polyP (p700) (Cat#EUI002, Kerafast, Inc. Boston, MA) was used in all assays. The polyP stock was prepared according to the manufacturer’s instructions in sterile water to a concentration of 102 mg/ml polyP, and this stock was diluted in cultures to make 15 µg/ml polyP. Bacterial survival assay To determine the effect of the inhibitors on the survival of E. coli , Mtb , Legionella , or Listeria in macrophages, human macrophages (from blood monocytes cultured with GM-CSF or M-CSF for 6 days) were infected with E. coli , Mtb , Legionella , or Listeria , in the absence or in the presence of 15 µg/ml polyP, 10 µg/ml ScPPX, or the indicated concentration of the inhibitor. At day 7, after removing loosely adhered cells as described above for PBMCs purification, 100 µL RBCSG (for E. coli , Legionella, and Listeria survival assay) or RBCSGLP (RBCSG containing 50 μg/mL leucine and 50 μg/mL pantothenate for Mtb survival assays) containing the indicated concentrations of the inhibitor without GM-CSF or M-CSF were added to macrophages in each well in type 353072, 96-well, tissue-culture-treated, polystyrene plates (Corning) and incubated for 30 minutes at 37 °C. polyP, ScPPX, or the inhibitor when assayed, was present in all incubation steps up to the Triton lysis step. Meanwhile, 1 mL of E. coli , Mtb , Legionella , or Listeria from a log phase culture was washed twice with RBCSG (for E. coli , Legionella and Listeria ) or RBCSGLP (for Mtb ) without GM-CSF or M-CSF by centrifugation at 12,000 x g for 2 minutes in a microcentrifuge tube, resuspended in 1 mL of RBCSG (for E. coli , Legionella and Listeria ) or RBCSGLP (for Mtb ), and the 600 nm optical density of 100 µl of the culture was measured with a Synergy Mx monochromator microplate reader (BioTek, Winooski, VT). 100 µl of RBCSG (for E. coli , Legionella and Listeria ) or RBCSGLP (for Mtb ) was used as a blank. The bacteria were diluted to an optical density of 0.5 (∼0.33 x 10 7 Legionella / ml; ∼0.766 x 10 7 Listeria / ml; ∼10 7 Mtb / mL) in RBCSG (for E. coli , Legionella and Listeria ) or RBCSGLP (for Mtb ). Mtb (∼1 µl), Legionella (∼3.3 µl), or Listeria (∼1.3 µl) was added to macrophages in each well such that there were ∼5 bacteria per macrophage considering ∼20% of the blood monocytes converted to the macrophages in the presence of GM-CSF or M-CSF ( 82 ). The bacteria-macrophage co-culture plate was spun down at 500 x g for 3 minutes with a Multifuge X1R Refrigerated Centrifuge (Thermo Scientific, Waltham, MA) to synchronize phagocytosis of the bacteria, and incubated for 2 hours at 37 °C. The supernatant medium was removed by gentle pipetting and was discarded. 100 µL of PBS warmed to 37 °C was added to the co-culture in each well, cells were gently washed to remove un-ingested extracellular bacteria, the PBS was removed, and 100 µL of RBCSG (for E. coli , Legionella, and Listeria ) or RBCSGLP (for Mtb ) with M-CSF or GM-CSF containing 200 µg/mL gentamicin (Sigma, St. Louis, MO) was added to the cells to kill the remaining un-ingested bacteria. After 2 hours, cells were washed twice with PBS as above to remove gentamicin. RBCSG (for E. coli , Legionella, and Listeria ) or RBCSGLP (for Mtb ) (100 µL) with M-CSF or GM-CSF was then added to the cells. After 4 and/or 48 hours of infection, macrophages were washed as above with PBS, the PBS was removed, and cells were lysed using 200 µL 0.1% Triton X-100 (Alfa Aesar) in PBS for 5 minutes at room temperature by gentle pipetting, and 20 µl and 100 µL of the lysates were plated onto agar plates (as described above for Mtb culture). The Mtb containing agar plates were incubated for 3 to 4 weeks or until the Mtb colonies appeared, whereas E. coli containing agar plates were incubated for 1 day (as described above for E. coli culture), Legionella containing agar plates were incubated for 3 days (as described above for Legionella culture), and Listeria containing agar plates were incubated for 2 days (as described above for Listeria culture). Bacterial colonies obtained from plating 20 µl and 100 µl lysates were manually counted, the number of viable ingested bacterial colonies per 20 µl and 100 µl lysates was calculated, and the number of viable ingested bacteria colony-forming units (cfu) per ml of lysate was then calculated, which correspond to the number of viable ingested bacteria in ∼2 x 10 5 macrophages. To calculate the percent of control, cfu/ml of the control of each independent experiment was considered 100%. To determine the effect of the inhibitors on the survival of Mtb in human macrophages for more than 48 hours, macrophages were infected with Mtb as described above, in the absence or in the presence of the indicated concentrations of the inhibitor. The indicated concentrations of the inhibitor were then additionally added to the cells at 24 and 48 hours after Mtb infection. At 72 hours (3 days of infection), macrophages were lysed and plated onto agar (as described above for lysates from 4 and 48 hours). The agar plates were incubated for 3 to 4 weeks or until the Mtb colonies appeared. Mtb colonies obtained from plating 20 µl and 100 µl lysates were manually counted, the number of viable ingested Mtb colonies per 20 µl and 100 µl lysates was calculated, and the number of viable ingested Mtb colony forming units (cfu) per ml of lysate was then calculated, which correspond to the number of viable ingested Mtb in ∼2 x 10 5 macrophages. To calculate the percent of control, cfu/ml of the control was considered 100%. Bacterial growth assay To investigate the effect of the inhibitors on bacterial growth, Mtb , Legionella, or Listeria bacteria were grown in a well of a 96-well, tissue culture-treated plate (# 353072, Corning) containing a final OD 600 of 0.1 in 200 µl of respective growth media as described above. OD600 was measured using a BioTek Synergy Mx monochromator microplate reader. Mtb , Legionella, or Listeria bacteria were incubated in the absence or in the presence of 10 µg/ ml ScPPX or the indicated inhibitor concentrations. Control wells contained medium with water or DMSO, which was similarly serially diluted in media. The plates were subsequently incubated in a container with humidity provided by wet paper towels at 37 °C in a humidified incubator. The OD 600 of the cells was measured daily for 6 days (for Mtb ) or at 12, 24, 48, 72, and 144 hours (for Legionella ) or at 6, 12, 24, and 48 hours (for Listeria ), and the bacterial growth curves were generated. Fluorescence microscopy Mtb survival assays were performed in type 353219 96 well, black/clear, tissue culture-treated glass bottom plates (Corning) with 10 5 cells/ well in 100 µl as described above. In a control experiment, uninfected macrophages were incubated for 24 hours. At 24 hours, macrophages with ingested Mtb were fixed with 200 µl of 4% paraformaldehyde/PBS for 10 minutes. Cells were washed 2 times with 200 µl of PBS and permeabilized with 200 µl of 0.1 % Triton X-100 (Alfa Aesar, Tewksbury, MA) in PBS for 5 minutes. Macrophages were washed twice with 200 µl of PBS, blocked with 200 µl of 1 mg/ ml type 0332 bovine serum albumin (BSA) (VWR) in PBS for 1 hour, and washed once with 200 µl of PBS. 200 µl of 1:2000 rabbit anti- Mtb antibody (Cat# OBT0947; Bio-Rad), 1:1000 rabbit anti-CD54 (Cat#67836; Cell Signaling, Danvers, MA), 1:1000 rabbit anti-CD206 (Cat#91992; Cell Signaling) antibody in PBS/ 0.1 % Tween 20 (Fisher Scientific, Pittsburgh, PA) (PBST) was added to macrophages and incubated at 4 °C overnight. Cells were washed three times with PBST and incubated with 200 µl of 1:500 Alexa 488 donkey anti-rabbit (711-546-152, Jackson Immunoresearch) or 1:500 Alexa 488 donkey anti-mouse (715-545-150, Jackson Immunoresearch) and 10 µg/ ml of DAPI in PBST for 1 hour. Cells were washed three times with 200 µl of PBST, and 200 µl of PBS was then added to the well. Each washing step was done for 5 minutes, and all steps were performed at room temperature if not indicated otherwise. Images of macrophages were taken with a 100× oil-immersion objective or 40× objective on a Ti2 microscope (Nikon), and deconvolution of images was done using the Richardson-Lucy algorithm ( 83 ) in NIS-Elements AR software. Figures were prepared using Microsoft PowerPoint. Immunoblot analysis Macrophages from the fluorescence microscopy assay described above, after 24 hours, were lysed in sample buffer (50 mM Tris/HCl, pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate [SDS] [J.T. Baker], 1% β-mercaptoethanol [Sigma], and 0.02% Bromophenol blue [Matheson]) containing 1x Pierce Protease Inhibitor, EDTA-Free (Cat# A32965, ThermoFisher) and heated to 95°C for 5 minutes. The samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE) using Tris-glycine 4-20% polyacrylamide gels (Lonza), transferred to a polyvinylidene fluoride blotting membrane (GE Healthcare), and immunoblotted according to the manufacturers’ instructions. Blots were blocked with 5% skim milk (BD) in PBS. The primary antibodies, diluted 1:1000 in PBST, were rabbit anti-LC3A/B (D3U4C) XP (Cat#12741, Cell Signaling) and mouse anti-GAPDH (Cat#60004-1-Ig, Proteintech, Rosemont, IL). The secondary antibodies were HRP-conjugated goat anti-rabbit IgG (Cat#111-035-144, Jackson ImmunoResearch) and HRP-conjugated goat anti-mouse IgG (Cat#115-035-146, Jackson ImmunoResearch), both diluted 1:2500 in PBST. Bound antibodies were detected using an enhanced chemiluminescence Western blotting kit (Thermo Fisher). Band intensities on the blots were quantified using ImageJ. Phagosome acidification assay To determine the effect of inhibitors on polyP-mediated inhibition of phagosome acidification in human macrophages, macrophages derived from blood monocytes cultured with GM-CSF or M-CSF for 6 days in type 353219, 96-well, black/clear, tissue-culture-treated, glass-bottom plates (Corning) with 10 5 cells per well in 100 µL (as described above) were incubated with a pH-sensitive pHrodo red labeled yeast (Cat# P35364, Thermo Fisher Scientific, Waltham, MA) (yeast hereafter), and the acidification of the yeast containing phagosomes was monitored as previously described ( 25 ). pHrodo red labeled yeast is non-fluorescent outside the cell but fluoresces brightly red in acidic phagosomes ( 49 ). 100 nM of Concanamycin A (ConcanA) (Cat#C9705-25UG, Sigma), a vacuolar-type H+-ATPase inhibitor that blocks phagosome acidification ( 50 ), was used as a positive control for inhibition of phagosome acidification. Proteasome activity assay At 24 hours of phagocytosis, as described for the bacterial survival assay, cultures of macrophages in the absence or in the presence of 15 µg/ml polyP, 4 nM Mg132 (Cat#474787, Sigma), or Mtb without or with 1000 nM inhibitor in type 353219, 96-well, black/clear, tissue-culture-treated, glass-bottom plates (Corning) were incubated with 100 µl/well of proteasome assay loading solution provided in a Proteasome Activity Kit (Cat#MAK172, Sigma, St Louis, MO). The plate was incubated in the dark for 1 hour, and proteasome activity was measured using a microplate reader following the manufacturer’s instructions. Metabolic activity assay Metabolically active cells transform the non-fluorescent blue dye resazurin into a fluorescent pink product resorufin ( 47 ). At 24 hours of phagocytosis, as described for the bacterial survival assay, macrophages in the absence or in the presence of Mtb , Legionella, Listeria, or inhibitor in 96-well, tissue culture-treated plates (# 353072, Corning) were incubated with prewarmed Deep Blue Cell Viability resazurin dye (Cat#424702, BioLegend, San Diego, CA) to a final concentration of 10% in each well ( 84 ). Plates were incubated at 37 °C for 12 hours. The fluorescence signal was then measured using a microplate reader following the manufacturer’s protocol. Statistical analysis Statistical analyses were performed using Prism 10 (GraphPad Software, Boston, MA) or Microsoft Excel. p < 0.05 was considered significant. Contact for Reagent and Resource Sharing Further information and requests for reagents may be directed to, and will be fulfilled by, the authors Ramesh Rijal ( ramesh.rijal{at}usm.edu ) or Richard Gomer ( rgomer{at}tamu.edu ). Author Contributions R. 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