P. aeruginosa disrupts ARP-2/3 mediated apical F-actin organization to induce intestinal deformation in C. elegans

P. aeruginosa disrupts ARP-2/3 mediated apical F-actin organization to induce intestinal deformation in C. elegans | 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 P. aeruginosa disrupts ARP-2/3 mediated apical F-actin organization to induce intestinal deformation in C. elegans View ORCID Profile AN Divyashree , View ORCID Profile Tanushree Sinha , View ORCID Profile Anup Padmanabhan doi: https://doi.org/10.1101/2025.10.08.681298 AN Divyashree 1 Department of Biology, Trivedi School of Biosciences, Ashoka University , Sonipat, Haryana India -131029 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for AN Divyashree Tanushree Sinha 1 Department of Biology, Trivedi School of Biosciences, Ashoka University , Sonipat, Haryana India -131029 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tanushree Sinha Anup Padmanabhan 1 Department of Biology, Trivedi School of Biosciences, Ashoka University , Sonipat, Haryana India -131029 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anup Padmanabhan For correspondence: anup.padmanabhan{at}ashoka.edu.in Abstract Full Text Info/History Metrics Supplementary material Preview PDF SUMMARY Apico-basal polarity is crucial for maintaining cortical actin organization and regulating secretory functions in intestinal epithelium. The opportunistic pathogen, Pseudomonas aeruginosa , exploits the interplay between cell polarity and cytoskeletal machineries to subvert host defenses. Mechanistic insights into such pathogen-induced cytoskeletal alterations have largely been derived from in-vitro epithelial monolayers, which may not capture the influences of multicellular physiology such as tissue mechanics, and innate immunity. Here, we show that the extracellular P. aeruginosa disrupts apical polarity in C. elegans enterocytes, leading to fragmentation of ARP-2/3 clusters and disorganization of apical F-actin. This disruption causes shedding of actin-rich vesicles into lumen and a pronounced apical deformation. Inhibition of CDC-42-ARP-2/3-mediated actin polymerization or PI3K-AKT signalling attenuated enterocyte deformation and extended the lifespan of C. elegans upon P. aeruginosa exposure. Our findings reveal a conserved strategy by which P. aeruginosa exploits cellular polarity machinery to disrupt host actin organization during extracellular infection of the intestinal epithelium. INTRODUCTION Constantly exposed to a diverse micro-organisms, the intestinal epithelium acts as a frontline sentinel for immune surveillance, detecting perturbations caused by dysbiosis, distinguishing harmless commensals from pathogens, and mounting context-appropriate immune response. During infection, bacterial pathogens elicit a wide range of responses from intestinal epithelial cells - some orchestrated by the pathogens to promote their survival and dissemination, and others initiated by the host to limit damage and trigger innate immunity. Unlike professional immune cells, enterocytes lack canonical pathogen recognition receptors (PRRs) that detect microbe-associated molecular patterns (MAMPs). Instead, they respond to pathogen effector activity and the disruptions they cause to physiological processes and cellular homeostasis, collectively termed “patterns of pathogenesis”. These include damage associated molecular patterns (DAMPs) triggered by pathogen effectors. Studies in model organisms such as Drosophila melanogaster and Caenorhabditis elegans (C. elegans) have highlighted the role of effector-triggered, cell autonomous surveillance in epithelial tissue ( Stuart et al , 2013 ; Tse-Kang et al , 2025 ; Vance et al , 2009 ). C. elegans, a bacterivorous soil nematode, inhabits microbe-rich environments and encounters a broad spectrum of microorganisms, including pathogens ( Berg et al , 2016 ; Schulenburg & Félix, 2017 ). While a protective cuticle ( Gravato-Nobre et al , 2005 ) and sophisticated pathogen-avoidance behaviours ( Lei et al , 2024 ; Melo & Ruvkun, 2012 ; Pereira et al , 2020 ; Prakash et al , 2021 ; Singh & Aballay, 2019b ; Zhang et al , 2005 ) limits pathogen exposure, the intestinal epithelium remains the primary site of infection due to the worm’s bacterial diet. Ingested bacteria are mechanically ground in the pharynx before entering the lumen, yet some pathogens survive, proliferate, and colonize the intestine. The C. elegans intestine is composed of 20 polarized epithelial cells arranged longitudinally, with apical surfaces facing the central lumen and basolateral surface engaged in cell-cell and cell-matrix adhesions ( Dimov & Maduro, 2019 ; McGhee, 2007 ). The apical surface is enriched in microvilli, providing the primary site for host-pathogen interaction ( MacQueen et al , 2005a ) ( Fig. 1A ). Polarity is maintained by structural and signalling complexes: the Crumbs complex (CRB-1, CRB-3, MAGUK-2) and the PAR complex (comprising PAR-3, PAR-6, PKC-3 kinase and CDC-42 GTPase) localize apically, while the Scribble complex (LET-413, LGL-1, and DLG-1) and PAR-1 kinase localize basolaterally. Lipids are also polarized, with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 or PIP 2 ] enriched apically and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 or PIP 3 ] basolaterally. This polarity is reinforced by polarity complexes, cytoskeletal organization, membrane trafficking, and junctional assemblies. ( Armenti & Nance, 2012 ; Bossinger et al , 2004 ; Legouis et al , 2000 ; Winter et al , 2012 ). Membrane polarity is also essential for the spatial localization and activation of Rho family GTPases ( Jaffe & Hall, 2005 ). In particular, CDC-42 activates the ARP-2/3 complex to promote branched actin polymerization, essential for the assembly and maintenance of apical microvilli ( Bernadskaya et al , 2011 ; Ma et al , 1998 ; Martin-Belmonte et al , 2007 ; Shafaq-Zadah et al , 2012 ; Thuenauer et al , 2022 ). During infection many pathogens employ diverse virulence strategies to subvert the host polarity and actin cytoskeletal machinery to alter cellular shape and mechanics ( Ruch & Engel, 2017 ). This facilitates intracellular entry and spread, or extracellular effector-induced inflammation and tissue damage ( Bastounis et al , 2022 ; Colonne et al , 2016 ). Conversely, epithelial cells may actively reorganize actin as part of their defense response. Thus, plasma membrane polarity-actin cytoskeleton cross talk is vital for epithelial integrity during infection ( Fleiszig et al , 1997 ; Tran et al , 2014 ). Download figure Open in new tab Figure 1: P. aeruginosa infection deforms the apical surface of intestinal epithelium in C. elegans (A) Schematic illustration of C. elegans intestinal epithelium depicting different regions of the enterocyte. (B) Representative Kaplan-Meier survival plot of N2 worms infected with P. aeruginosa (PA14-magenta) or E. coli (OP50-grey) at 25⁰ C. (n = 100 worms, N=2) (C) Medial confocal sections of C. elegans intestines expressing mCherry::ACT-5 localized to apical surface. While lumen distension increases with age in animals exposed to with P. aeruginosa or E. coli (red double headed arrows), intestinal surface deformation is seen only upon exposure to with P. aeruginosa (red filled arrowheads), but not E. coli (blue ROI; blue filled arrowheads). with P. aeruginosa infection also led to actin vesicles budding of apical surface (Red ROI; red arrows). (D) Bar graphs showing increased percentage of intestinal deformation with increase in the duration of exposure to with P. aeruginosa . (E) Schematic showing the estimation of deformation index ( δ apical ) of apical surface (F) Representative violin plot showing quantification of deformation index of apical surface shown in Fig.1C . (N=3; n= 10 worms per treatment per set). (G) Plot showing progressive deformation of apical surface of intestinal lumen from individual worms measured over 3 days following P. aeruginosa or E. coli exposure. (n=5) (H) Medial confocal images of C. elegans expressing fluorescently labelled apical markers upon exposure to P. aeruginosa or E. coli . ERM-1::eGFP (apical regions containing microvilli), PAR-6::mNeonGreen (apical PAR polarity complex), IFB-2::wScarlet (intermediate filaments), PGP-1::GFP (apical plasma membrane). Scale bar is 20 µm. P-values were calculated by Mann-Whitney U test. ****p<0.0001, ***p <0.001, and * p <0.05 The opportunistic pathogen, Pseudomonas aeruginosa a major cause of nosocomial infections and infects a wide range of hosts, including C. elegans ( Balla & Troemel, 2013 ; Mahajan-Miklos et al , 1999 ; Tan et al , 1999a ). In worms, P. aeruginosa infection induces diverse stress responses, mitotic quiescence, and apoptosis ( Kuss-Duerkop & Keestra-Gounder, 2020 ; Pellegrino et al , 2014 ; Dunbar et al , 2012 ; McEwan et al , 2012 ; Bollen et al , 2024 ). In the absence of professional immune cells, C. elegans upon detecting infection, activates innate immune signalling such as the TIR-1-p38 MAP kinase and the DAF-2/DAF-16 insulin/IGF-1 pathways ( Peterson et al , 2023 ). DAF-2 dependent phosphorylation of PI3K/AGE-1 recruits AKT-1/AKT-2 kinases that in turn phosphorylate DAF-16. This kinase cascade results in cytoplasmic retention of DAF-16, thereby regulating gene expression ( Ewbank, 2006 ). DAF-16 is known to induce expression of antimicrobial genes, detoxifying enzymes and bacterial avoidance ( Balla & Troemel, 2013 ; Cohen & Troemel, 2015 ; Irazoqui et al , 2010 ; Peterson et al , 2022 ; Pukkila-Worley & Ausubel, 2012 ). Avoidance behaviour is also triggered by mechanical cues, such as intestinal distension (“bloating”), mediated by TRPM channels ( Filipowicz et al , 2021 ; Kumar et al , 2019 ; Singh & Aballay, 2019b , 2019a ). Here, we investigated the mechanism underlying cytoskeletal remodelling and polarity disruption following P. aeruginosa infection. We find that P. aeruginosa exploits PI3K-AKT pathway to perturb CDC-42-ARP-2/3-mediated actin organization in C. elegans enterocytes. The resultant disruption in epithelial polarity and discharge of actin rich vesicles from the apical surface locally alters the surface mechanics resulting in a deformed intestinal lumen surface that is associated with decreased organism viability. RESULTS Pseudomonas aeruginosa infection results in C. elegans intestinal deformation Consistent with previous studies, exposure to P. aeruginosa strain PA14 resulted in reduced C. elegans survival (TD PA14 ∼3 days) compared to E. coli (OP50) (TD OP50 ∼8 days) ( Fig. 1B ). This lethality has been partly attributed to the colonization of the intestinal lumen by P. aeruginosa ( Tan et al , 1999a ). To examine the impact of P. aeruginosa infection on host cytoskeletal organization, we imaged C. elegans intestines expressing mCherry fused to the N-terminus of ACT-5, the intestine-specific actin isoform ( MacQueen et al , 2005b ; Szumowski et al , 2016 ). In E. coli -fed worms, ACT-5 appeared as a smooth parallel lining along the intestinal lumen ( Fig. 1B and 1C ; blue arrowhead, OP50). Despite age related intestinal distension ( Egge et al , 2019 ; Irazoqui et al , 2010 ; Singh & Aballay, 2019a ), the ACT-5 signal remained smooth even after 3 days on E. coli ( Fig.1C ; red double-headed arrows in OP50/day-3, and S1A). Although lumen was distended following P. aeruginosa exposure ( Fig. 1C , red double-headed arrows in PA14 day-3, and Fig S1A), apical surface characteristics were markedly distinct. In contrast to the smooth surface seen in E. coli -fed animals, P. aeruginosa -fed worms displayed extensive wrinkles and surface deformations ( Fig. 1C ; PA14,filled red arrowheads), with vesicle-like structures budding outward from the deformed apical surface ( Fig. 1C ; PA14, red arrows in enlarged ROI). Compared to only ∼4% (2/54) of E. coli -fed worms, ∼48%(27/56) of P. aeruginosa -exposed worms showed such deformations after 1-day ( Fig. 1C ). By day-3, the incidence rose to ∼83% (43/52) in P. aeruginosa -fed worms compared to ∼19% (10/52) on E. coli ( Fig. 1D ). Magnitude of intestinal deformation depends on the duration of P. aeruginosa infection To quantify changes in intestinal morphology, we defined a dimensionless “apical deformation index” ( δ apical ), as the ratio of apical lumen length to total worm length ( Fig. 1E ). E. coli -fed worms consistently maintained ‘ δ apical OP50 =1’, whereas P. aeruginosa exposure resulted in ‘ δ apical PA14 >1’, with values increasing over time, indicating progressive deformation ( Fig. 1F ). Tracking individual worms revealed that those fed with E. coli , the value of δ OP50 remained ∼1, but in worms exposed to P. aeruginosa , deformation was evident by day-1 and worsened over subsequent days ( Fig. 1G ). In acute infection assays, 12-hour exposure did not cause significant deformation, whereas 24-hour exposure induced noticeable apical distortion even after transfer back to E.coli (Fig. S1B). To test whether deformation extended beyond the apical surface, we examined transgenic worms co-expressing LET-413::mCherry and DLG-1::GFP, the Scribble and Discs Large orthologs in C. elegans, as markers for basolateral surface and adherens junctions (AJ), respectively ( Bossinger et al , 2004 ; Legouis et al , 2000 ; McMahon et al , 2001 ; Riga et al , 2021 ). Neither AJ positioning nor basal deformation index ( δ basal ) was significantly altered by P. aeruginosa exposure ( Fig. 5A and S1C). However, lateral surfaces showed LET-413 enrichment and altered morphology ( Fig. 5A and 5C ), indicating that deformation was restricted to apical and lateral domains. Next we confirmed that the intestinal deformation was independent of oocytes or uterine expansion following fertilization by examining males (Fig. S1D and S1E). F-actin staining using Phalloidin-AlexaFluor647 and LifeACT::mRuby expression showed visible apical deformation specifically upon P. aeruginosa exposure, eliminating the possibility that the infection phenotype might be an artifact of mCherry::ACT-5 transgene expression (Fig. S1F and S1H, red filled arrowheads). Finally, we validated the phenotype using additional apical/sub-apical markers: PGP-1::GFP, ERM-1::GFP, PAR-6::GFP, and IFB-2::GFP, that localized to apical membrane, microvillar structure or subapical positions, respectively ( Fig. 1H ) ( Geisler et al , 2020 ; Göbel et al , 2004 ; Sepers et al , 2022 ). In all cases, P. aeruginosa exposure resulted in clear deformation of the apical surface topology. Slow killing’ assay, typically uses nematode growth media (NGM) containing 0.35% peptone ( Tan et al , 1999a ). To minimize any confounding effects from elevated peptone concentrations on intestinal epithelial response, we conducted our infections on standard NGM containing 0.25% peptone, optimised for C. elegans growth ( Stiernagle, 2006 ). We observed intestinal deformation in worms infected on 0.35% peptone media indistinguishable from 0.25% peptone (Fig. S1I and S1J). Our lifespan assays mirrored previously reported kinetics using 0.35% peptone-containing NGM ( Figure 1A ) ( Tan et al , 1999a ). Thus we confirmed that our infection conditions faithfully recapitulate chronic P. aeruginosa infection, leading to progressive apical and lateral intestinal deformation. CDC-42 mediates Pseudomonas -induced intestinal deformation Members of RHO family GTPases, primarily RHO-1, RAC-1, and CDC-42, are key upstream regulators of F-actin assembly and organization, that co-ordinate actin dynamics in a spatiotemporally controlled manner ( Ann Mack & Georgiou, 2014 ; Jaffe & Hall, 2005 ). P. aeruginosa -infecting MDCK cells disrupt RHO signalling through effector proteins that interfere with GTPase-activating proteins (GAPs), leading to the disorganization of actin architecture ( Cowell et al , 2005 ; Kazmierczak & Engel, 2002 ; Krall et al , 2000 ; Pederson et al , 1999 ; Sun & Barbieri, 2004 ). To test whether P. aeruginosa -induced surface deformation in C. elegans enterocytes might similarly depend on RHO GTPase-mediated actin re-organization, we individually depleted RHO-1, CED-10 (RAC-1), and CDC-42 in adult worms expressing intestinal mCherry::ACT-5, and subsequently exposed them to P. aeruginosa or E. coli . As in the RNAi control, exposure to P. aeruginosa resulted in apical deformation in rho-1(RNAi) or ced-10(RNAi) animals ( Fig. 2A , S2A and S2B). In contrast, cdc-42(RNAi) worms exposed to P. aeruginosa displayed no deformation ( Fig. 2A and 2B ). This lack of deformation was not due to impaired pathogen sensing in cdc-42(RNAi) worms (Fig. S2D and S2E) ( Estes et al , 2010 ). Consistent with its role in suppressing intestinal deformation, cdc-42(RNAi) worms exhibited significantly extended lifespan (8 days) compared to RNAi controls (4 days) upon P. aeruginosa exposure ( Fig. 2C ). Download figure Open in new tab Figure 2: Exposure to P. aeruginosa results in cytoplasmic mis-localization of CDC-42, ARX-2 and ACT-5 (A) RNAi mediated depletion of cdc-42 , but not rho-1 or ced-10 rescues the PA-14 induced intestinal deformation. Red filled arrowheads arrow heads show deformed regions of the lumen, while red open arrow heads denote the smooth intestinal lumen surface in cdc-42(RNAi) worms. (B) Quantification showing reduced deformation index of intestines in cdc-42(RNAi) worms compared to L4440 (control) upon P. aeruginosa exposure for two days. Measurements are plotted as violin plot indicating median and interquartile of each data set. (N = 3; n=27)) (C) Representative Kaplan-Meier survival plot of cdc-42(RNAi) or L4440 (control) worms subjected to P. aeruginosa or E. coli infection. CDC-42 depletion partially rescues P. aeruginosa induced shortening of lifespan (TD50 cdc42(RNAi) = 8 days, TD50 control = 4 days) (N=2; n= 50 worms per set and per condition) (D, G and J) Medial cross-sections of intestinal lumen of C. elegans expressing (D) CDC-42::GFP, (G) ARX-2::TagRFP, and (J) mCherry::ACT-5 following exposure to E. coli or P. aeruginosa . Images shown are pseudo-colored to highlight changes in the intensity. Warm colors represent higher intensity and lower intensity by the dimmer colors. Cyan arrows indicate the cytoplasmic enrichment of these proteins in animals infected with P. aeruginosa . Where visible, nuclei of the intestinal cells are marked with letter ‘n’. Scale bar -20 µm. (E and F) Violin plots showing cytoplasmic (E) and apical (F) intensities of CDC-42::GFP upon P. aeruginosa or E. coli infection. Measurements are mean ±95% C.I. Each dot represents one animal and the data shown is the cumulation of three independent experiments (N=3; n=33). (H and I) Violin plots indicating cytoplasmic (E) and apical (F) intensities of ARX-2::TagRFP upon P. aeruginosa or E. coli infection. Measurements are mean ±95% C.I. Each dot represents individual worm (N=2; n≥21) (K and L) Violin plots of cytoplasmic (E) and apical (F) intensity measurements of mCherry::ACT-5 upon P. aeruginosa or E. coli infection. Measurements are mean ±95% C.I. Each dot represents individual worm (N=2; n≥14) Scale bar is 20 µm. P-values were calculated by Mann-Whitney U test. ****p<0.0001, ***p <0.001, **p <0.01and * p <0.05. P. aeruginosa infection leads to cytoplasmic accumulation of G-actin The absence of intestinal deformation in cdc-42(RNAi) worms prompted us to investigate the intracellular localization of CDC-42 and its downstream effector, the actin nucleating ARP-2/3 complex, during P. aeruginosa infection. We used worms co-expressing ARX-2::TagRFP (an essential subunit of the ARP-2/3 complex) at its endogenous locus, and CDC-42::GFP, expressed exogenously under the native cdc-42 promoter ( Neukomm et al , 2014 ; Wu et al , 2017 ). In control animals maintained on E. coli , both CDC-42 and ARX-2 localized predominantly to the apical membrane of the enterocytes, with little accumulation in the cytoplasm over 2 days of exposure ( Fig. 2D and 2G ; OP50 panel and Fig. S2C). Upon exposure to P. aeruginosa , both cytoplasmic and apical CDC-42 levels increased markedly ( Fig. 2D enlarged ROI, 2E and 2F). Cytoplasmic levels of ARX-2 also increased (∼2.6 fold) ( Fig. 2H ) though apical ARX-2 remained unchanged relative to E. coli ( Fig. 2I ). Intracellular actin mirrored that of ARX-2, with ∼2.5 fold increase in cytoplasmic levels but none in apical levels ( Fig. 2J - 2L ). Surprisingly, Phalloidin-AlexaFluor647 staining and LifeACT::mRuby did not show any cytoplasmic staining, suggesting that the accumulated cytoplasmic actin pool is primarily G-actin (Fig. S1F-S1H). Thus, the increased cytoplasmic levels following P. aeruginosa infection reflects accumulation of G-actin rather than loss of F-actin from the apical surface. P. aeruginosa -induced disruption of cortical actin results in apical surface deformation It seemed counterintuitive that apical ARX-2 and ACT-5 intensities did not increase despite CDC-42 enrichment. To clarify this conundrum, we measured apical levels of ACT-5 and ARX-2 in cdc-42(RNAi) worms, and found them to be significantly reduced (see OP50 panels in Figs. 3A, S3A, S3B and S3D). Thus apical actin and ARP-2/3 complex are sensitive to CDC-42-depletion, but not to P. aeruginosa -induced CDC-42-enrichment. The absence of deformation in cdc-42(RNAi) animals could therefore reflect a direct effect of CDC-42 depletion or an indirect effect via reduced ARP-2/3-mediated F-actin polymerization. To distinguish these, we depleted ACT-5 or ARX-2 in worms co-expressing CDC-42::GFP and ARX-2::TagRFP. Due to act-5(RNAi) -associated developmental defects, analyses were restricted to day 1 post-infection. Strikingly, act-5(RNAi) and arx-2(RNAi) worms, like cdc-42(RNAi) showed rescue of deformation phenotype following infection despite elevated CDC-42 levels (red open arrowheads in Fig.3C -E,3G and S3E). Download figure Open in new tab Figure 3: Inhibition of ARP-2/3 dependent actin polymerization prevents P. aeruginosa -induced apical surface deformation (A) Intestinal lumen marked by mCherry::ACT-5 from control and cdc-42(RNAi) animals exposed to P. aeruginosa or E. coli . Red arrowheads indicate rescue of intestinal deformation in cdc-42(RNAi) animals. (B) Cytoplasmic ACT-5 intensity quantified from intestinal epithelium of control and cdc42(RNAi) worms, day 2 post infection. Quantifications are mean ±95% C.I. (n ≤ 14). (C) Confocal images of intestinal epithelium from control and arx-2(RNAi) animals co-expressing CDC-42::GFP and ARX-2::tagRFP, and subjected to E. coli and P. aeruginosa infection. All images are from 2-days post infection. Open red arrows indicate rescue of the intestinal deformation. (D) Quantification of cytoplasmic CDC-42::GFP in control and arx-2(RNAi) animals following 2-day exposure to P. aeruginosa or E. coli . The violin plot shows the distribution of data with median and interquartile for each data set. p-values n≥8 for all the sets. expect n= 5 for PA14 arx-2 (RNAi) . (E) Violin plot showing apical deformation index quantification for arx-2(RNAi) animals exposed to E. coli or P. aeruginosa . Plot shows the median and interquartile of each data set. (n ≤ 11). (F) Medial plane image of intestinal lumen co-expressing PGP1::GFP and mCherry::ACT5 subjected to control (L4440) and arx-2 RNAi followed by E. coli and Pseudomonas exposure. All the images are from one day post E. coli and Pseudomonas exposure. (G) Medial plane image of intestinal lumen co-expressing CDC42::GFP and ARX2::TagRFP subjected to control (L4440, empty vector) and act-5 RNAi followed by E. coli and Pseudomonas exposure. All the images are from one day post E. coli and Pseudomonas exposure. Open red arrow marks represent rescue of the intestinal deformation. unless otherwise specified, Scale bar is 20 µm. P-values were calculated by Mann-Whitney U test. ****p<0.0001, ***p <0.001, and * p <0.05. To probe the function of ARP-2/3 dependent actin polymerization in P. aeruginosa -induced intestinal deformation, we analysed worms co-expressing mCherry::ACT5 and an apical membrane maker, PGP-1::GFP. In control worms fed with E. coli , ACT-5 and PGP-1 were uniformly co-localized at the apical surface ( Fig. 3F ; control panel). In arx-2(RNAi) intestines, apical actin was absent and instead appeared as cytoplasmic aggregates, even when fed with E. coli ( Fig. 3F ; arx-2(RNAi) red filled arrowheads). This ruled out cytoplasmic actin accumulation as a possible cause of P. aeruginosa -induced deformation. ARP-2/3 disruption has been implicated in cytoplasmic mislocalization of membrane bound components such as DLG-1 and ERM-1 ( Bernadskaya et al , 2011 ). PGP-1 displayed diffused apical localization in arx-2(RNAi) intestines ( Fig. 3F ; arx-2(RNAi) red hollow arrowheads). These results suggested the central role of ARP-2/3 polymerized actin in maintenance of apical membrane organization. Importantly, CDC-42 and ARX-2 maintained apical localization even in the absence of actin ( Fig. 3F and 3G ), confirming their upstream role in the actin polymerization pathway. Collectively, these results demonstrate that rescue of apical deformation in cdc-42(RNAi) and arx-2(RNAi) intestines following P. aeruginosa exposure was due to disruption of ARP-2/3 mediated actin polymerization. Fragmented ARP-2/3 clustering and F-actin disorganization drive release of membrane vesicles into the intestinal lumen following P. aeruginosa exposure Although overall intensities of apical ARX-2 and ACT-5 were comparable between worms exposed to P. aeruginosa and E. coli ( Fig. 2I and 2L ), their surface localization patterns were markedly distinct. In E. coli -fed worms, ARX-2 was evenly distributed at the apical surface ( Fig. 4A ; OP50, cyan arrowhead) essential for contiguous actin polymerization, supporting brush border integrity ( Bidaud-Meynard et al , 2021 ; MacQueen et al , 2005b ; Stutz et al , 2015 ). In contrast, P. aeruginosa -infected animals exhibited fragmented ARX-2 clusters, interspersed with ARX-2-depleted regions ( Fig. 4A ; PA14, white arrows). We sought to correlate the cortical actin distribution with this fragmented ARP-2/3 localization using worms co-expressing mCherry::ACT-5 and ARX-2::GFP. Medial confocal sections of intestines from this strain when exposed to P. aeruginosa resulted in apical actin to protrude as a ‘bud-like’ structure into the lumen, emerging from ARX-2-free apical surface ( Fig 4B ; actin protrusions - yellow arrowheads, fragmented ARP-2/3 – cyan arrows and Fig. 1C ; red arrows in enlarged ROI). By day-2, these protrusions pinched off as vesicles, coinciding with apical surface deformation ( Fig. 1C ; day-2 PA14 panel - red arrows in enlarged ROI and Fig. 4D -cyan arrows in enlarged ROI). Lattice light sheet microscopy of the intestinal lumen surface of animals expressing mCherry::ACT-5 exposed to P. aeruginosa expressing GFP revealed extracellular pinching of actin rich vesicles from the apical surface into the lumen ( Fig. 4C ; enlarged ROI, cyan arrows). Download figure Open in new tab Figure 4: Fragmented ARP-2/3 disrupts actin assembly leading to extracellular vesicle release from apical surface (A) Intensity visualization of medial confocal images of C. elegans intestines expressing ARX-2::TagRFP showing a fragmented distribution of ARX-2 (cyan arrows) on day-2 post P. aeruginosa exposure. ARX-2 shows a contiguous localization in worms exposed to E. coli (cyan arrowheads). Surface intensity of plots of enlarged ROIs highlights the fragmentation. Scale bar -10 µm. (B) C. elegans co-expressing ARX-2::GFP and mCherry::ACT-5 showing actin structures (yellow arrowheads) permeating into the lumen from ARX-2-free regions (cyan arrows). Scale bar -10 µm. (C) Lattice light sheet images acquired 2 days post exposure of PA14-GFP or OP50-GFP -fed C. elegans expressing intestinal mCherry::ACT5. Images were acquired in 13.3X objective and represented as maximum intensity projection in volume mode. Cyan arrows in enlarged ROI indicate the vesicles emerging from apical surface into the lumen. (D) Membrane bound actin rich vesicles bud off from intestinal apical surface following 2-day P. aeruginosa exposure (cyan arrows in ROI). Enlarged ROI shows vesicles at various stages of budding – (a) initial bulging, (b) bud formation, and (c) released vesicle. An intensity linescan drawn across the extracellular vesicle indicates actin (magenta) surrounded by the apical plasma membrane marked by PGP-1::GFP (green). Scale bar -5 µm. (E) LifeACT::mRuby localizes to F-actin in the apical vesicles (cyan arrow) of C. elegans intestines exposed to Pseudomonas . Scale bar -10 µm. P-values were calculated by Mann -Whitney U test. ****p<0.0001, ***p <0.001, and * p <0.05. To determine whether these extracellular actin structures were membrane-enclosed, we examined worms co-expressing mCherry::ACT-5 and PGP-1::GFP. Medial confocal images on day-2 post-infection revealed apical buds at various stages: initial protrusion ( Fig. 4D ; ROIs 1a & 2a), membrane pinching (1b & 2b), and free vesicles in the lumen (cyan arrows, 1c & 2c). Intensity line-scan analysis confirmed that these vesicles consisted of cortical actin cores surrounded by PGP-1::GFP-labelled apical membrane. LifeACT::mRuby labeled these vesicles, indicating filamentous actin content ( Fig. 4E ). We estimated the vesicle diameter to be ∼ 0.59 µm (Fig. S4A). These results support the view that P. aeruginosa exposure led to disordered ARP-2/3 localization at the apical lumen surface destabilising cortical actin organization, resulting in the release of F-actin rich vesicles from the apical surface into lumen. P. aeruginosa infection disrupts apical polarity of enterocyte epithelium Fragmented ARP-2/3 clustering and vesicle shedding suggested a loss of apical membrane identity. P. aeruginosa attachment to MDCK cells triggers a transient PI(3,4,5)P 3 accumulation at the apical surface, resulting in the loss of apical membrane identity ( Kierbel et al , 2007 , 2005 ; Thuenauer et al , 2022 ). To check if P. aeruginosa might cause a similar disruption to the polarity in C. elegans intestinal epithelium, we analysed the localization of the C. elegans Scribble homolog, LET-413 and the cell junction marker, DLG-1. The LET-413 complex and DLG-1 have been reported to be essential for maintenance of basolateral domains in C. elegans by regulating the localization of apical polarity components ( Bossinger et al , 2004 ; McMahon et al , 2001 ). In E. coli -fed worms, the localization of LET-413 was limited to the basolateral membrane with the apical surface largely devoid of any LET-413 ( Fig. 5A ; OP50 panel, basolateral surface -yellow filled arrowhead, apical surface -yellow asterisks) ( Legouis et al , 2000 ). Upon exposure to P. aeruginosa, we observed a significant increase in LET-413 levels along the apical and lateral membranes ( Fig. 5A ; PA14 panel, apical surface - yellow arrows, lateral membrane-yellow open arrowhead, 5B and 5C), indicating disruption in cell polarity. P. aeruginosa infection did not affect the position of AJ( Fig. 5A ; cyan arrows). The absence of intestinal deformation in cdc-42(RNAi) animals prompted us to investigate if CDC-42 might also regulate apico-lateral polarity following P. aeruginosa exposure. To this end, we checked the localization of LET-413 in control (L4440) and cdc-42(RNAi) worms. The enhanced apical localization of LET-413 in the intestines of control (L4440) worms was significantly diminished in cdc-42(RNAi) animals following P. aeruginosa exposure ( Fig. 5D ). Conversely, we did not detect any disturbance to the apical localization of CDC-42 and ARP-2/3 in let-413(RNAi) intestines, in agreement with previous reports indicating that LET-413 depletion has minimal effect on epithelial polarity (Fig S5A) ( Riga et al , 2021 ). Moreover, similar to control animals, P. aeruginosa exposure of let-413(RNAi) worms resulted in apical surface deformation as well as elevated cytoplasmic levels of CDC-42 (S5A and S5B). Taken together, these results suggest that upon exposure to P. aeruginosa, the apical surface C. elegans intestine acquires basolateral characteristics in a CDC-42-dependent mechanism. PI3K-AKT signalling is required for CDC-42 mis-localization and intestinal deformation Disruption of the PI3K-AKT pathway impedes P. aeruginosa infection in MDCK cells ( Kierbel et al , 2005 ). Similarly, long-lived C. elegans mutants such as akt-1 , akt-2 and age-1 /PI3K exhibit resistance to P. aeruginosa ( Evans et al , 2008 ; Garsin et al , 2003 ) suggesting a conserved host response to P. aeruginosa infection between mammalian epithelium and C. elegans enterocytes. To examine the conservation in cellular response to P. aeruginosa between mammals and C. elegans , we diminished PI3K-AKT signalling by a simultaneous depletion of AKT-1 and AKT-2 using RNAi and checked the intestinal localization of LET-413, CDC-42 and ARX-2. Depletion of AKT-1/AKT-2, like in cdc-42(RNAi) worms, prevented the apical localization of LET-413 following P. aeruginosa infection ( Fig. 5D ). Evidence for crosstalk between CDC-42 dependent actin assembly and PI3K-AKT in mammalian cells prompted us to measure CDC-42 and ARX-2 levels in the intestines of akt-1(RNAi)+akt-2(RNAi) and age-1(RNAi) animals following P. aeruginosa exposure ( McCormick et al , 2019 ; Yang et al , 2012 ). Intestinal epithelium of animals depleted of AKT or AGE-1 displayed diminished apical and cytosolic ARX-2 localization compared to control animals ( Fig. 5E , S5D - S5G), and also did not display apical deformation following P. aeruginosa exposure ( Fig. 5F ). Our results, taken together with earlier studies on MDCK cells ( Kierbel et al , 2007 ) suggests that PI3K dependent apical enrichment of PI(3,4,5)P 3 following exposure to P. aeruginosa , disrupts apical membrane polarity and de-localizes ARP-2/3 dependent actin assembly from the apical surface resulting in intestinal deformation. These findings re-enforces the importance of polarity disruption as a key event following P. aeruginosa infection and prior to intestinal deformation. Download figure Open in new tab Figure 5: P. aeruginosa induced polarity disruption is mediated via PI3K-AKT kinase pathway (A) LET-413 shows enhanced accumulation in the apical (yellow arrow) and lateral (yellow arrowhead) surfaces C. elegans enterocytes upon P. aeruginosa infection. DLG-1 marks cell junctions (cyan arrows). Double asterisks shows absence of LET-413 at the apical surface in intestines of C. elegans exposed to E. coli . (B and C) Quantification of (B) apical, and (C) lateral LET413 levels in enterocytes of worms following E. coli (OP50) or P. aeruginosa (PA14) infection. Each data point represents individual worm. Measurements are plotted as violin plot indicating median and interquartile of each data set. (n=12). (D) Depletion of AKT-1 and AKT-2, or CDC-42 rescues P. aeruginosa induced loss of polarity. In contrast to control (L4440; yellow asterisks), apical recruitment of LET-413 in response to P. aeruginosa infection is absent in akt-1/akt-2(RNAi) and cdc-42(RNAi) (yellow asterisks). (E) Disruption of PI3K-AKT pathway abrogates P. aeruginosa induced mis-localization of CDC-42, ARX-2 and actin. Medial confocal plane of intestinal lumen co-expressing CDC-42::GFP and ARX-2::TagRFP subjected to akt-1/akt-2 and age-1 RNAi followed by E. coli and Pseudomonas exposure for two days. Closed yellow arrow indicate intestinal deformation. Yellow asterisks denote rescue of deformation. (F) Violin plot showing apical deformation index( δ apical ) quantification for akt-1/akt 2 and age-1(RNAi) animals exposed to E. coli or P. aeruginosa for two days. Plot shows the median and interquartile of each data set (N =2, n≥22). Scale bar in 20 µm. P-values were calculated by Mann -Whitney U test. ****p<0.0001, ***p <0.001, and **p <0.01 Extracellular localization of Pseudomonas is sufficient to induce intestinal deformation Pseudomonas aeruginosa functions both as an extracellular and intracellular pathogen infecting a wide range of hosts ( Resko et al , 2024 ). Transient activation of PI3K pathway resulting in apical membrane acquiring basolateral characteristics was resulted in internalization of P. aeruginosa by MDCK cells ( Kierbel et al , 2005 ). Electron microscopy studies of P. aeruginosa -infected C. elegans intestine have also reported Pseudomonas invasion into enterocytes ( Irazoqui et al , 2010 ; Xue et al , 2024 ). To determine whether intestinal deformation in C. elegans results from P. aeruginosa breaching the epithelial barrier, we performed live confocal imaging of infection using GFP-expressing E. coli or P. aeruginosa . Unlike E. coli (OP50-GFP), which were in very few numbers even after two days of exposure, P. aeruginosa (PA14-GFP) cells formed visible aggregates throughout the intestinal lumen as early as one-day post infection ( Fig. 6A ). Interestingly, apical surfaces adjacent to these aggregates displayed mild contortions, which progressed into pronounced deformation by day 2 ( Fig. 6A ; red filled triangles in bottom row-PA14). Orthogonal reconstruction (YZ plane) of confocal (XY) planes revealed that PA14-GFP localization remained restricted to the lumen with no detectable signal in the cytoplasm of the intestinal cells ( Fig.6A ; PA14 panel). These observations were confirmed using lattice light-sheet microscopy ( Fig. 4C and Supplementary Movie S1). To test whether apical surface deformation is a general enterocyte response to any pathogen, we conducted parallel infection assays using Salmonella enterica serovar typhimurium (Stm), a Gram-negative bacterium known to modulate CDC-42 activity during mammalian infections ( Bandyopadhyay et al , 2024 ) and cause lethality in C. elegans ( Fig. S6A) ( Aballay et al , 2000 ; Desai et al , 2019 ). Stm-GFP infection in worms expressing mCherry::ACT-5, resulted in efficient intestinal colonization by day 2 similar to P. aeruginosa (Fig. S6B). However, despite this robust lumen colonization, in contrast to P. aeruginosa , Stm-exposed intestines did not display any surface deformation ( Fig. 6A ; middle row, yellow open arrowheads). Taken together, these results indicate that the intestinal deformation is a pathogen-specific response by the C. elegans enterocyte to extracellular Pseudomonas infection. Download figure Open in new tab Figure 6: Extracellular P. aeruginosa can induce intestinal deformation in C. elegans (A) Confocal medial images of intestinal epithelium from C. elegans expressing ACT ::mCherry and exposed to GFP expressing E. coli (top panel), S. typhymurium (middle panel) and P. aeruginosa (bottom panel) for 3-days post infection. Dashed lines indicate cross-sectional plane whose orthogonal view is presented adjacent to the image. Red hollow ( E. coli ) and yellow ( S. typhimurium) arrowheads indicate intestinal distension (bloating), but smooth lumen surface. Red filled arrowheads indicate the deformation of the intestine in worms infected with P. aeruginosa . (B) Medial confocal images of intestines from C. elegans expressing mCherry::ACT-5 animals grown on heat killed E. coli (top panel) and P. aeruginosa (bottom panel). Hollow red arrow indicates the absence of intestinal deformation upon exposure to heat killed P. aeruginosa . (C) Medial confocal section of intestinal epithelium expressing mCherry::ACT5 exposed to 12 h of bacterial supernatant as indicated. Closed red arrows indicates intestinal deformation. (D) Medial confocal images of intestines from C. elegans expressing mCherry::ACT-5 exposed to E. coli (OP50) (top panel), P. aeruginosa (PA14) (middle panel) and P. aeruginosa (PA14ΔgacA) (bottom panel). Filled red arrows indicates intestinal deformation. (E) Quantification of deformation index ( δ apical ) of the apical intestinal surface in C. elegans expressing intestinal mCherry::ACT-5 exposed to E. coli (OP50), P. aeruginosa (PA14) and P. aeruginosa. Each data point represents individual animal and measurements are plotted as violin plot indicating median and interquartile of each data set. (n =10) (F) Kaplan-Meier survival plot of worms described in Fig. 6A upon exposure to E. coli (OP50), P. aeruginosa (PA14) and GacA deleted P. aeruginosa (PA14Δ gacA ). Worms were grown on E. coli till L4 stage and transferred on to the respective infection plates. (n = 50 worms, N=2) (G) Schematic of the proposed mechanism for P. aeruginosa induced intestinal apical surface deformation in C. elegans . Scale bar in 20 µm. P-values were calculated by Mann -Whitney U test. ****p<0.0001, ***p <0.001, and * p <0.05 Physical association between live P .aeruginosa and the intestinal surface is not essential for apical deformation Given the key role for physical association with host cells in P. aeruginosa pathogenesis (via various adhesins, including flagella, type IV pili, type III secretion system (T3SS), and lectins ( Bucior et al , 2012 ; Comolli et al , 1999 ; Thuenauer et al , 2022 ), we asked whether a direct attachment of P. aeruginosa to the apical membrane of C. elegans intestine was necessary to trigger tissue deformation. First, we tested if exposure to live microbes is essential for inducing deformation of apical surface, by exposing animals to heat-killed E. coli or P. aeruginosa instead of live bacteria. Since worms displayed no noticeable defects in growth, maturation or egg laying, we concluded that heat-killed bacteria as food source was not nutritionally limiting. However, unlike live P. aeruginosa , heat-killed P. aeruginosa failed to induce intestinal deformation ( Fig. 6B ; open red triangle). Our observation agrees with previous reports showing heat-killed P. aeruginosa does not elicit immune responses in C. elegans ( Irazoqui et al , 2010 ). Since extracellular P. aeruginosa induces intestinal deformation, we hypothesized that secreted factors may mediate this effect. To test this, we exposed worms to cell-free filter-sterilized supernatant collected from P. aeruginosa or E. coli cultures. Remarkably, a 12-hour exposure to P. aeruginosa supernatant alone was sufficient to trigger apical surface deformation in the C. elegans intestine ( Fig. 6C ). The global response regulator, GacA governs the expression of numerous virulence factors secreted by P. aeruginosa, including toxins and components in biofilm regulation ( Barta et al , 1992 ; Kitten et al , 1998 ; Parkins et al , 2001 ; Reimmann et al , 1997 ) and has also been implicated in inducing developmental defects and activating the unfolded protein response (UPR) in C. elegans intestinal epithelium ( Pellegrino et al , 2014 ). To assess the role of GacA in apical surface deformation, we exposed mCherry::ACT-5-expressing worms to E. coli , P. aeruginosa or a gacA deletion mutant (PA14Δ gacA ) strain. The intestines of worms exposed to PA14Δ gacA exhibited an intermediate level of intestinal deformation compared to those infected with wild-type PA14 ( Fig. 6D and 6E ). Importantly, a gacA deletion mutant (PA14Δ gacA ) retains partial pathogenicity, causing ∼50% lethality in infected worms ( Fig. 6F ) ( Feinbaum et al , 2012 ; Tan et al , 1999b ). These results suggest that while GacA partially contributes to P. aeruginosa pathogenesis in C. elegans , a GacA-independent mechanism also exists, through which the pathogen elicits tissue surface deformation. Taken together, these results support the view that while intestinal distension is a general tissue response to microbial colonization of the lumen, mechanical deformation of apical surface is a specific response to extracellular heat-labile component from P. aeruginosa . DISCUSSION Here we report that P. aeruginosa exposure induces two distinct morphological alterations in the C. elegans intestine; (a) lumenal distension, reflected by increased intestinal width, and (b) deformation of the apical surface, seen as wrinkling of the lumen. While lumen distension has been previously described as a general response to microbial colonisation, we show that apical surface deformation is specific to P. aeruginosa exposure. P. aeruginosa infection in MDCK cells recruit PI3K to the attachment sites, causing PIP 3 enrichment, apical mis-localization of basolateral proteins, and disruption of epithelial polarity through perturbation of the Rho GTPases - RHO-1, RAC-1 and CDC-42 ( Aktories, 2011 ; Kazmierczak & Engel, 2002 ; Kierbel et al , 2007 ; Krall et al , 2000 ). Consistent with this, we find that inhibition of either PI3K-AKT signalling or CDC-42-dependent actin assembly diminished both intestinal deformation and pathogen-induced lethality. P. aeruginosa infection also led to apical mis-localization of the basolateral marker LET-413, indicating a loss of apical membrane identity, though no apical markers were mis-localized basolaterally. The rescue observed in cdc-42(RNAi) animals further implicated CDC-42 in pathogen-induced actin remodelling. However, depletion of the downstream polarity factor, LET-413, failed to rescue deformation, suggesting that CDC-42 contributes primarily via actin assembly rather than polarity regulation. Supporting this, depletion of ACT-5 or disruption of ARP-2/3 phenocopied cdc-42(RNAi) . In line with previous reports ( Kierbel et al , 2005 ; Paradis & Ruvkun, 1998 ), we noted that attenuation of PI3K-AKT signalling rescued the intestinal deformation by limiting ARP-2/3 mis-localization. Thus, manipulations that prevent deformation were also found to extend survival, supporting a strong correlation between intestinal cell surface mechanics and organism viability. These results highlights the central role of CDC-42-ARP-2/3-mediated actin polymerization in P. aeruginosa- induced deformation. In uninfected animals, ARP-2/3, together with actin-binding proteins such as PLST-1 and FLN-2, forms evenly distributed apical clusters ( Bidaud-Meynard et al , 2021 ). ARP-2/3 polymerizes contiguous F-actin to support microvillar structures ( Fig. 4A ). P. aeruginosa infection fragmented these clusters, disrupting local actin organization, and destabilized the apical surface, promoting vesiculation of actin-rich membrane into the lumen. Because apical actin structures in enterocytes show minimal mobility, their disruption cannot be rapidly replenished, leading to progressive surface destabilization ( Bernadskaya et al , 2011 ; Bidaud-Meynard et al , 2021 ) ( Fig. 6G ). This is consistent with electron micrographs showing actin protrusions in MDCK cells ( Kierbel et al , 2005 ) and reports of microvillar loss and host debris accumulation in the C. elegans lumen during P. aeruginosa infection ( Irazoqui et al , 2010 ; Xue et al , 2024 ). Intriguingly, depletion of ARX-2 or ACT-5 prevented vesicle release and surface deformation, suggesting that a uniform depletion of ARP-2/3 or actin reduces the availability of F-actin for vesiculation. A similar protective effect was observed in MDCK cells, where Latrunculin A-mediated actin depolymerization attenuated P. aeruginosa infection by up to 60-fold ( Kazmierczak et al , 2004 ). CDC-42 upregulation and activation have been reported in polarized MDCK cells ( Kazmierczak et al , 2004 ). While P. aeruginosa infection in C. elegans enterocytes led to cytoplasmic accumulation of CDC-42, ARP-2/3 and actin, we suspect the accumulated CDC-42 and ARP-2/3 pools to be largely inactive, given that the cytoplasmic actin is primarily G-actin. In mammals, RHO-mediated actin regulation and homeostasis are monitored by pyrin- and NOD-dependent pathways, but no equivalent surveillance has been reported in C. elegans ( Mostowy & Shenoy, 2015 ). Although cytoplasmic actin accumulation correlated with infection, it did not directly cause deformation. Whether it may be sensed as a pattern of pathogenesis remains to be addressed. Moreover, MAPK signalling remained intact in cdc-42(RNAi) animals following infection, indicating that CDC-42 depletion does not impair microbial sensing. P. aeruginosa is generally considered as an extracellular pathogen and its intracellular lifestyle is usually associated with tissue injury. While P. aeruginosa has been shown to infiltrate the cytoplasmic space in many host systems, we have not observed epithelial breach in our experiments in the C. elegans intestine. The C. elegans epithelium, similar to a fully confluent monolayer of polarized MDCK cells, is refractory to intracellular P. aeruginosa infection. The surface deformation phenotype could, however, be recapitulated by exposure to cell-free supernatant from P. aeruginosa cultures but not by heat-killed bacteria suggested the role of a heat-labile factor. Given the extracellular lifestyle of P. aeruginosa in polarized epithelia, extracellular membrane vesicles (OMVs) rich in surface components, quorum-sensing molecules, toxins and other virulence factors, are plausible mediators of deformation signal ( Bauman & Kuehn, 2009 , 2006 ; Kuehn & Kesty, 2005 ; Mashburn & Whiteley, 2005 ). Thus extracellular P. aeruginosa exploits PI3K-AKT activation to perturb ARP-2/3-mediated actin organization, leading to the loss of apical polarity and cytoskeletal integrity in C. elegans enterocytes. Our findings reveal an evolutionarily conserved strategy by which P. aeruginosa subverts actin regulation during extracellular infection. We speculate that pathogen-mediated disruption of polarity impairs apically directed transport and secretion of antimicrobial peptides, and surface-binding lectins. An attenuated antimicrobial response could allow bacterial proliferation in the lumen and a rapid spreading of the bacteria in the population. AUTHOR CONTRIBUTIONS D.A.N and A.P conceived the project. D.A.N, T.S and A.P designed the experiments. D.A.N and T.S performed experiments. D.A.N and T.S analysed the data with input from A.P. A.P wrote the manuscript with input from D.A.N and T.S. Graphical Abstract Download figure Open in new tab ACKNOWLEDGEMENTS This work was supported by DBT-Wellcome India Alliance Fellowship (IA/I/18/1/503624) and ANRF Core research grant (CRG/2023/004638) to A.P, Department of Biotechnology Junior research Fellowship (DBT/2024-25/AshokaUni/2486) to T.S., and core funding support from the Trivedi School of Biosciences, Ashoka University. We acknowledge the infrastructure support from the Central Bio-imaging facility and Ashoka-Zeiss Core Imaging Facility at Ashoka University. We thank Ms. Megha Rai for help with vesicle size quantification, Ms. Shraddha Nirmal for technical assistance and Ms. Purna Pardeshi for some of the illustrations. We are grateful to the labs of Arnab Mukhopadhyay, Anindya Ghosh-Roy, Emily Troemel, Grégoire Michaux, Jogender Singh, Mike Boxem, Mahak Sharma and Varsha Singh, for sharing C. elegans and bacterial strains. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40OD010440). We tare grateful to Ishani Sharma, Saravanan Palani and Jogender Singh for their feedback on the manuscript. Funder Information Declared Wellcome Trust/DBT India Alliance , IA/I/18/1/503624 Department of Science and Technology, https://ror.org/03k93wc57 , CRG/2023/004638 Department of Biotechnology , DBT/2024-25/AshokaUni/2486 Footnotes The revised version consists of additional experiments to support the claims in this manuscript. References 1. ↵ Aballay A , Yorgey P & Ausubel FM ( 2000 ) Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans . Curr Biol 10 : 1539 – 1542 OpenUrl CrossRef PubMed Web of Science 2. ↵ Aktories K ( 2011 ) Bacterial protein toxins that modify host regulatory GTPases . Nat Rev Microbiol 9 : 487 – 498 OpenUrl CrossRef PubMed 3. ↵ Ann Mack N & Georgiou M ( 2014 ) The interdependence of the Rho GTPases and apicobasal cell polarity . Small GTPases 5 : 37 – 41 OpenUrl 4. ↵ Armenti ST & Nance J ( 2012 ) Adherens junctions in C. elegans embryonic morphogenesis . 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Share P. aeruginosa disrupts ARP-2/3 mediated apical F-actin organization to induce intestinal deformation in C. elegans AN Divyashree , Tanushree Sinha , Anup Padmanabhan bioRxiv 2025.10.08.681298; doi: https://doi.org/10.1101/2025.10.08.681298 Share This Article: Copy Citation Tools P. aeruginosa disrupts ARP-2/3 mediated apical F-actin organization to induce intestinal deformation in C. elegans AN Divyashree , Tanushree Sinha , Anup Padmanabhan bioRxiv 2025.10.08.681298; doi: https://doi.org/10.1101/2025.10.08.681298 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cell Biology Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41911) Biophysics (21436) Cancer Biology (18578) Cell Biology (25482) Clinical Trials (138) Developmental Biology (13371) Ecology (19887) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15599) Genomics (22483) Immunology (17728) Microbiology (40364) Molecular Biology (17163) Neuroscience (88537) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15129) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9817) Zoology (2269)