Mechanistic Insights into Human Antimicrobial Peptide-Induced Activation of a Broadly Conserved Bacterial Signaling System

Mechanistic Insights into Human Antimicrobial Peptide-Induced Activation of a Broadly Conserved Bacterial Signaling System | 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 Mechanistic Insights into Human Antimicrobial Peptide-Induced Activation of a Broadly Conserved Bacterial Signaling System View ORCID Profile Samuel A. Adeleye , View ORCID Profile Abraham F. Mechesso , Arpita Mukherjee , View ORCID Profile Guangshun Wang , View ORCID Profile Srujana S. Yadavalli doi: https://doi.org/10.1101/2025.05.06.652532 Samuel A. Adeleye 1 Waksman Institute of Microbiology and Department of Genetics, Rutgers University , Piscataway, New Jersey 08854 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Samuel A. Adeleye Abraham F. Mechesso 2 Department of Pathology and Microbiology, College of Medicine, University of Nebraska Medical Center , Omaha, Nebraska 68198 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Abraham F. Mechesso Arpita Mukherjee 1 Waksman Institute of Microbiology and Department of Genetics, Rutgers University , Piscataway, New Jersey 08854 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Guangshun Wang 2 Department of Pathology and Microbiology, College of Medicine, University of Nebraska Medical Center , Omaha, Nebraska 68198 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Guangshun Wang For correspondence: sam.yadavalli{at}rutgers.edu gwang{at}unmc.edu Srujana S. Yadavalli 1 Waksman Institute of Microbiology and Department of Genetics, Rutgers University , Piscataway, New Jersey 08854 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Srujana S. Yadavalli For correspondence: sam.yadavalli{at}rutgers.edu gwang{at}unmc.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Antimicrobial peptides (AMPs) represent a promising class of therapeutics against bacterial pathogens. While their direct bactericidal mechanisms are well-characterized, how bacteria sense and respond to these peptides at sublethal concentrations remains poorly understood. Here, we investigate the activation of the Escherichia coli PhoQ-PhoP signaling system by the human antimicrobial peptide LL-37 and its derivatives (KR-12 and RI-10). We demonstrate that these peptides exhibit variable antimicrobial potency but surprisingly similar abilities to activate the PhoQ-PhoP pathway, indicating that signaling function is separable from bactericidal activity. Notably, sublethal concentrations of these peptides induce significant cell elongation, a phenotype dependent on PhoQ and mediated by the upregulation of QueE, which interferes with bacterial cell division. Contrary to the previous model suggesting peptides activate PhoQ passively by displacing its inhibitor MgrB, we observed enhanced cell elongation in ΔmgrB strains across all tested peptides, including RI-10, lacking antibacterial activity. Our findings suggest peptides actively stimulate PhoQ through a mechanism independent of MgrB dissociation, providing a more refined understanding of the peptide signaling through the PhoQ-PhoP system. These insights into bacterial adaptation mechanisms against host-derived peptides may guide the development of peptide therapeutics with enhanced efficacy against drug-resistant pathogens. Significance statement Antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics, yet how bacteria detect and respond to these host defense molecules remains poorly understood. This study investigates the interaction between bacterial sensing systems and AMPs at the genetic and molecular levels. Unlike other eukaryotes with multiple cathelicidin peptides, humans have only one cathelicidin that produces various active fragments through processing. Rather than creating multiple detectors, E. coli deploys an elegant solution, the PhoQ-PhoP signaling system that recognizes the conserved antimicrobial region shared by all LL-37 fragments. We demonstrate that peptides directly stimulate PhoQ independent of their bactericidal activity, even in the absence of inhibitor MgrB, inducing cell elongation. These insights may inform the development of effective peptide-based therapeutics against drug-resistant pathogens. Introduction When exposed to specific cationic antimicrobial peptides (AMPs) at sublethal concentrations, Escherichia coli activates the PhoQ/PhoP signal transduction pathway—a conserved two-component system crucial for stress adaptation in Gram-negative bacteria ( 1 , 2 ). The PhoQ/PhoP signaling system is also an important pathway for adaptation and survival in conditions such as low Mg 2+ , mildly acidic pH, and hyperosmolarity in E. coli and closely related enterobacteria ( 3 , 4 ). Activated PhoQ-PhoP regulate numerous genes, including those responsible for remodeling the outer membrane, activation of efflux pumps, and proteolytic degradation of the peptides that help bacteria resist cationic AMPs ( 5 – 8 ). Strong activation of the PhoQ-PhoP system is observed when cells are treated with a human platelet-derived AMP (C18G), causing the upregulation of QueE, an enzyme primarily known for its role in the queuosine biosynthetic pathway that modifies specific tRNAs ( 2 ). Remarkably, elevated QueE levels lead to the accumulation of this protein at the bacterial division septum, where it interacts with the cell division machinery. This interaction disrupts normal septation processes downstream of Z-ring formation, resulting in inhibited cell division and the development of filamentous bacterial morphology. This mechanism represents a sophisticated bacterial stress response that temporarily halts proliferation when facing host antimicrobial challenges. Human cathelicidin, LL-37, is one of the best-studied AMPs ( 9 – 11 ) with a broad spectrum of activity against bacteria, fungi, parasites, viruses, and cancer cells ( 11 – 15 ). LL-37 is known to activate the PhoQ-PhoP system and exert its antimicrobial action primarily by interacting with bacterial membranes, inserting itself into the lipid bilayer, and leading to membrane destabilization and the formation of pores ( 1 , 16 – 18 ). This disruption results in the loss of membrane integrity, causing leakage of cellular contents and ultimately leading to bacterial cell death. LL-37’s efficacy against pathogens is influenced by the composition of the growth medium, particularly the presence of high salt concentrations and divalent cations ( 19 ). In our current understanding, cationic antimicrobial peptides interact with the acidic patch in the periplasmic sensing domain of PhoQ, displacing divalent cations (particularly Mg 2+ ) that normally repress PhoQ activity, leading to PhoQ autophosphorylation and subsequent phosphorylation of PhoP ( 1 ). Besides peptides and other signals, a 47 amino acid long small membrane protein called MgrB interacts with and inhibits PhoQ by affecting its kinase activity ( 20 , 21 ). Specific residues in the transmembrane and periplasmic regions of MgrB are important for the interaction with PhoQ ( 22 ). It is proposed that the sensing of AMPs by PhoQ at physiological concentrations of magnesium is dependent on MgrB ( 23 ). A conformational change in PhoQ upon AMP binding is thought to disrupt the PhoQ-MgrB complex, resulting in PhoQ activation via derepression. This model suggests that AMPs play an inert role in stimulating PhoQ. Human cathelicidin LL-37 contains 37 amino acids and starts with a pair of leucine residues. Triple-resonance NMR structural studies established that the C-terminal tail of LL-37 (residues 33-37) is disordered and not required for membrane-targeting mediated antimicrobial activity ( 24 ). This led to the use of several derivatives of the LL-37 peptide from the central region for therapeutic development ( 11 , 24 , 25 ). KR-12 is a 12-amino acid-long, truncated peptide derived from human LL-37, which is toxic to bacteria but not human cells ( 24 ). It has been widely utilized in peptide engineering ( 26 ). To effectively develop AMPs as novel peptide therapeutics, it is imperative to study the bacterial response to these peptides, including the biochemical and regulatory pathways that underlie this process. While there has been tremendous progress in establishing the direct antimicrobial action of AMPs such as LL-37 against bacteria ( 27 ), much less is known about the phenotypic changes elicited by these peptides at sub-lethal doses and how their length and sequence contribute to bacterial response. In addition, it is unclear if AMPs have a direct and active role in stimulating PhoQ. To gain deeper insights into the stress response mechanism to AMPs in E. coli , we utilized LL-37, its shortest anti- E. coli peptide KR-12, and an even shorter non-antibacterial peptide RI-10, resulting from the truncation of the N-terminal lysine and C-terminal arginine of KR-12. In particular, we first examined the antibacterial activity of LL-37 and its smaller fragments, KR-12 and RI-10, using different E. coli strains under physiologically relevant conditions. We determined the killing kinetics and characterized the peptides using membrane permeabilization and depolarization assays. Next, we investigated whether LL-37 and its fragments, at concentrations below MIC, are effective in activating the PhoQ/PhoP signaling system by analyzing both the changes in cell morphology and PhoP-dependent transcriptional reporter activity as readouts. Finally, based on our analysis of cells lacking the PhoQ inhibitor, MgrB, we propose a model of how peptides like LL-37 and its derivatives may interact with bacterial histidine kinase and affect bacterial physiology. Our study sheds light on the molecular mechanism of bacterial detection of biologically relevant peptide LL-37, which is known to play a key role in human health via both antimicrobial and immune regulatory roles ( 9 – 12 , 27 ). Our results reveal that the bacterial PhoQ-PhoP two-component system detects a variety of LL-37 antimicrobial fragments in a specific manner by recognizing the consensus core antimicrobial region. Results Antibacterial activity of LL-37 and its derivatives against E. coli To better delineate the antibacterial activity of LL-37, KR-12, and RI-10 against E. coli , we compared both their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against four E. coli strains (MG1655, E423-17, E416-17, and 25922, Tables1 , S1 , S2 ) under three different media conditions: Mueller Hinton Broth (MHB), tryptic soy broth (TSB), or supplemented minimal A medium (MinA). Gentamicin was included as a non-peptide antibiotic control. LL-37 exhibited the highest potency with MIC values between 2 and 8 μM, depending on the media used ( Table 1 ). Interestingly, the KR-12 peptide, which is shorter than LL-37 by 25 amino acids, is also active against the E. coli isolates tested. The MIC and MBC values for KR-12 are similar or 2-4 fold higher than those obtained for LL-37. The values obtained in minimal medium were in a similar range as those obtained in other media, especially for E. coli K-12 MG1655 strain ( Tables 1 , S1 ). The shortest LL-37 variant, RI-10, did not show antibacterial activity against any of the isolates tested here across different media conditions, or after dilution of MHB to 10%. From here on, for characterization of the antibacterial and physiological effects of these peptides, we used the E. coli MG1655 strain grown in the supplemented minimal medium. Next, we investigated the time-dependent killing kinetics of LL-37, KR-12, and RI-10 to further substantiate their bactericidal properties using a concentration corresponding to 2x MIC values ( Figure S1 ). LL-37 (2× MIC) completely eradicated the exponential phase of E. coli K-12 MG1655 in 1 hour, while KR-12 caused about a 4-log fold reduction in 2 hours. As expected, RI-10 displayed no activity at the concentration tested. Together, these results show that full-length LL-37 and KR-12 exhibit antibacterial properties against E. coli but not RI-10. View this table: View inline View popup Download powerpoint Table 1. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values for LL-37 and its derivatives against E. coli K-12 MG1655. MIC values (µM) of antimicrobial peptide LL-37 and its derivatives KR-12, RI-10, and KE-10 against E. coli K-12 MG1655 in supplemented minimal medium (Min A), Mueller Hinton broth (MHB), and tryptic soy broth (TSB). The corresponding MBC values (µM) are shown within parentheses. Gentamicin is included as a bactericidal antibiotic control. Data represent values obtained from three independent biological replicates. LL-37 and KR-12, but not RI-10, disrupt bacterial membranes Given that LL-37 targets bacterial membranes, we wondered if the shorter peptide variants, KR-12 and RI-10, may retain and exhibit activity against E. coli membranes. To assess membrane disruption, we conducted membrane permeabilization and depolarization experiments. We analyzed membrane permeability by the uptake of a membrane-impermeable red dye, propidium iodide (PI), either in the presence of a peptide, 0.1% Triton-X (positive control) or PBS (untreated). Treatment with either LL-37 or KR-12 led to an increase in membrane permeability in a dose-dependent manner, although LL-37 was more effective ( Figure 1A-C ). To assay membrane depolarization, we used DiBAC 4 ( 3 ), a probe for membrane potential, whose fluorescence increases upon entering depolarized cells. As expected, LL-37 depolarized membranes well with increasing concentration of the peptide ( Figure 1D-F ). Similarly, KR-12 also caused membrane depolarization but to a lower extent relative to LL-37. Consistent with the data from MIC assay and killing kinetics, RI-10 did not have a discernible effect on membrane permeability and depolarization. Together, these results suggest that KR-12, like LL-37, targets the bacterial membrane as its mode of action. Download figure Open in new tab Figure 1. Membrane permeabilization (A-C) and depolarization (D-F) activity of a series of LL-37, KR-12, and RI-10 in E. coli K-12 MG1655. The assay was performed in supplemented MinA with 0.1 mM MgSO 4 . RFU: Relative fluorescence intensity. Data are represented as mean ± range for two independent replicates. Sub-MIC levels of LL-37 and KR-12 activate the PhoQ/PhoP signaling system and cause cell elongation Cationic antimicrobial peptides, LL-37 and C18G activate the PhoQP pathway in E. coli and related bacteria ( 1 , 2 ). In addition, E. coli cells elongate upon exposure to a sub-lethal dose of C18G, leading to filamentous growth ( 2 ). This filamentation is associated with conditions that strongly activate PhoQ/PhoP. We asked if the truncated LL-37 derivatives retain the ability to activate the PhoQP system. We used a promoter-reporter strain containing P mgrB - yfp to measure the transcriptional activity of the mgrB gene, whose expression is PhoQP-dependent. In parallel, we observed the cells by phase contrast microscopy to test if the peptides induce any changes in E. coli cell morphology. Our analysis revealed that there is a 3 to 3.5-fold increase in PhoQP-regulated transcriptional activity when cells are treated with a sub-MIC dose of LL-37 at 1.25 µM and KR-12 at 5 µM ( Figure 2A ). RI-10 showed weak activation of ∼1.7-fold higher than the untreated control. As the amino acid sequence of RI-10 (i.e., residues 19-28 of LL-37) is included in both KR-12 (residues 18-29) and LL-37, we also asked how a different LL-37 segment, KE-10 (residues 10-19), with a minimal sequence overlap with KR-12 and RI-10 ( Table S2 ), behaves. Like RI-10, KE-10 showed no antibacterial activity against E. coli (MIC > 64 µM in Table 1 ). In contrast to RI-10, the KE-10 peptide showed negligible activation of the PhoQP system based on the transcription activity measurement of the mgrB gene, implying that there is a requirement for a particular peptide sequence for this activation. Although LL-37 was long known to activate the PhoQP system, its impact on E. coli cell morphology, especially at concentrations below the MIC, has not been studied previously. Upon observation by phase contrast microscopy, both LL-37 and KR-12-treated cells displayed elongation with heterogeneous cell lengths ( Figure 2B ). The average cell lengths increased by ∼1.5-fold for cells exposed to LL-37 and KR-12 when compared to the control cells ( Figure 2C ). However, both RI-10 and KE-10 treated cells did not show a significant change in cell length ( Figure 2B-C ).Together, these data elucidate that LL-37, KR-12, and RI-10 are functional in stimulating the PhoQP pathway and eliciting a physiological response, but not KE-10. However, only full-length LL-37 and its minimal antibacterial peptide KR-12 are strong enough to cause morphological changes similar to what was observed for the C18G peptide previously ( 2 ), whereas two shorter LL-37 segments (RI-10 and KE-10), which lost antibacterial activity, were unable to. Download figure Open in new tab Figure 2. PhoQP-regulated transcription and cell morphology of E. coli cells treated with sub-MIC levels of antimicrobial peptides. (A) PhoQP-dependent promoter P mgrB -yfp reporter activity, (B) phase contrast micrographs, and (C) quantification of cell lengths of E. coli MG1655 (TIM92) cells grown in the presence of sub-MIC levels of antimicrobial peptides LL-37 (1.25 µM), KR-12 (5 µM), RI-10 (5 µM), and KE-10 (5 µM) in supplemented MinA with 0.1 mM MgSO 4 . Data are obtained from at least three biological replicates; mean and median values are shown in red text and black bars, respectively. The number of cells analyzed is indicated for each sample. P-values indicate the results of a Student’s t-test: ****P ≤ 0.0001, *P ≤ 0.05, and “ns” = P > 0.05. Scale bar = 5 µm. Cell elongation caused by sub-MIC levels of peptides is dependent on PhoQ and QueE To gain additional insight into the cell elongation caused by peptides, we examined the morphology of the cells upon deletion of phoQ . As expected, Δ phoQ cells did not elongate when treated with LL-37 and KR-12 ( Figure 3A ). There was no increase in PhoQP-dependent transcriptional activity of P mgrB , even in the presence of LL-37 and KR-12 in Δ phoQ cells ( Figure 3B ), and the average cell lengths were similar relative to the no-peptide control ( Figure 3C ), confirming that the cell elongation phenotype associated with these AMPs is dependent on PhoQ activation. In previous work, sub-MIC levels of C18G are shown to cause filamentation in wild-type E. coli cells, which is mediated by increased expression of a PhoP-dependent gene called queE , encoding a protein with dual activities in the inhibition of cell division and the synthesis of queuosine tRNA modification ( 2 , 28 ). We asked if the morphological phenotype observed here for cells exposed to LL-37 and KR-12 is also mediated by QueE or through an independent mechanism. Using a queE deletion strain, we carried out microscopy and measured cell lengths and transcriptional reporter activity. Δ queE suppressed the elongation phenotype observed when cells were treated with either LL-37 or KR-12 ( Figures 3A , 2B ). It is worth noting that Δ queE cells grown in the presence of these peptides still display an increase in PhoQP-regulated transcriptional reporter activity similar to the wild-type cells ( Figures 3D , 2A ). Consistently, Δ queE cells exposed to peptides did not show a difference in the average cell length relative to the controls (no peptide, KE-10, Figure 3E ). Overall, filamentation and heterogeneity in cell size observed upon treatment with LL-37 and KR-12 peptides are both PhoQ- and QueE-dependent. Download figure Open in new tab Figure 3. PhoQP-regulated transcription and cell morphology of ΔqueE and ΔphoQ cells treated with sub-MIC levels of antimicrobial peptides. (A) Phase contrast micrographs, (B, D) PhoQP-dependent promoter P mgrB -yfp reporter activity, and (C, E) quantification of cell lengths of ΔphoQ (TIM100) cells and Δ queE (SAM31) cells grown in the presence of sub-MIC levels of the antimicrobial peptides LL-37 (1.25 µM), KR-12 (5 µM), RI-10 (5 µM), and KE-10 (5 µM) in supplemented MinA with 0.1 mM MgSO 4 . Data are obtained from at least three biological replicates; mean and median values are shown in red text and black bars, respectively. The number of cells analyzed is indicated for each sample. P-values indicate the results of a Student’s t-test: ****P ≤ 0.0001, ***P ≤ 0.001, *P ≤ 0.05, and “ns” = P > 0.05. Scale bar = 5 µm. Deletion of PhoQ inhibitor, MgrB, increases cell responsiveness to LL-37 and its derivatives E. coli cells, carrying an mgrB deletion, show an overall, robust increase in PhoQP-controlled gene expression ( 20 ). These cells experience higher stimulation of PhoQ under magnesium-limiting conditions, similar to the strong activation of this system observed in wild-type cells grown in the presence of C18G at intermediate magnesium concentrations ( 2 ). Consequently, Δ mgrB cells grow as long filaments, and this phenotype is dependent on upregulation of QueE expression by PhoQ/PhoP ( 2 ). It is proposed that MgrB mediates the sensing of cationic AMPs by PhoQ at intermediate concentrations of magnesium, therefore, LL-37, KR-12, and RI-10 may activate PhoQ only when MgrB is present. In this case, we expect Δ mgrB cells to show no significant change in either the PhoP-dependent transcriptional reporter activity or cell morphology when treated with peptides vs. no treatment. We examined how LL-37 and its derivatives influence the activity of the PhoQP system and cell lengths in a Δ mgrB strain. As expected, in the absence of peptides, Δ mgrB cells showed an increase in P mgrB transcription relative to the wild-type cells under the same growth conditions ( Figures 4A-B ). When the AMPs were added to Δ mgrB cells, there was a modest increase of ∼1.4-fold in the PhoQP-regulated transcriptional reporter activity for LL-37, as observed previously using the same media conditions carrying 0.1 mM Mg 2+ ( Figure 4A ) ( 20 ). The addition of peptides KR-12, RI-10, and KE-10 did not lead to a substantial increase in reporter activity, suggesting that PhoP-mediated transcription is not sensitive in response to the two peptides in cells lacking MgrB. This result is consistent with a previous study where Δ mgrB cells did not show an increase in PhoP-regulated transcription in the presence of another AMP, C18G ( 23 ). Download figure Open in new tab Figure 4. PhoQP-regulated transcription and cell morphology of ΔmgrB cells treated with sub-MIC levels of antimicrobial peptides. (A) PhoQP-dependent promoter P mgrB -yfp reporter activity in ΔmgrB (AML20) cells, (B) comparison of PhoQP-dependent promoter P mgrB -yfp reporter activity in ΔmgrB (AML20) cells with that of WT E. coli (TIM92) from Fig. 2A , and (C) phase contrast micrographs of WT E. coli (TIM92) and ΔmgrB (AML20) cells, (D) quantification of cell lengths of ΔmgrB (AML20) cells, and (E) comparison of cell lengths in ΔmgrB (AML20) cells with that of WT E. coli (TIM92) from Fig. 2C . Cells were grown in the presence of sub-MIC levels of the antimicrobial peptides LL-37 (1.25 µM), KR-12 (5 µM), RI-10 (5 µM), and KE-10 (5 µM) in supplemented MinA with 0.1 mM MgSO 4 . Arrows in panel (E) indicate the PhoQ activation status. Data are obtained from at least three biological replicates; mean and median values are shown in red text and black bars, respectively. The number of cells analyzed is indicated for each sample. P-values indicate the results of a Student’s t-test: ****P ≤ 0.0001, **P ≤ 0.01, *P ≤ 0.05, and “ns” = P > 0.05. Scale bar = 5 µm. To investigate this further, we analyzed the morphology of Δ mgrB cells in the presence of peptides, wherein cell elongation is PhoQ-dependent and a direct function of strong PhoQP activation. As expected, we noticed filamentation in Δ mgrB cells treated with LL-37 and KR-12, but only short, typical cells in the KE-10-treated or no-peptide control cells, under this growth condition with 0.1 mM Mg 2+ ( Figure 4C ). It was intriguing, however, to see that RI-10-treated Δ mgrB cells also displayed elongation, suggesting that RI-10 may interact more effectively with PhoQ to activate it, in the absence of MgrB. In other words, loss of the inhibitor MgrB appears to unmask the effect of RI-10 peptide on PhoQ stimulation, resulting in an evident cell elongation phenotype. Interestingly, cell size quantification revealed greater heterogeneity and higher average cell lengths across all three peptides for Δ mgrB of 6-8 μm ( Figure 4D ). The mean cell lengths for LL-37, KR-12, and RI-10-treated Δ mgrB cells were higher than the corresponding values obtained for the wild type by 2.5-, 2.3-, and 1.9-fold, respectively; however, there is no difference in cell length in the KE-10-treated cells ( Figures 4D-E ), indicating a central role of the RI-10 containing LL-37 segments in activation in the absence of mgrB . It is known that high stimulation of PhoQ from exposure to LL-37 results in differential regulation of specific PhoP-regulated promoters, including that of mgrB ( 29 ). Also, upon activation by LL-37, a mutated mgrB-t7a promoter (with a T7A substitution in the PhoP-binding site) shows a higher fold change in transcriptional activity relative to the wild-type mgrB promoter. To substantiate whether there may be an increased transcriptional response to peptide treatment via this promoter, we analyzed wild-type and Δ mgrB strains carrying the mgrB-t7a promoter. We observed a significant increase in cell size heterogeneity in wild-type cells treated with LL-37 and KR-12, and Δ mgrB cells treated with LL-37, KR-12, and RI-10 ( Figure S2 ). The cells did not display an increase in cell length when treated with control peptide KE-10 or in the absence of a peptide. If the exacerbated cell elongation observed in peptide-treated Δ mgrB cells is indeed due to a PhoQP-dependent mechanism that is mediated by QueE, we should see this phenotype suppressed upon deletion of queE . Consistent with our hypothesis, Δ mgrB Δ queE cells treated with LL-37, KR-12, and RI-10 did not show an increase in PhoQ-dependent transcriptional reporter activity and displayed a loss of filamentation and a decrease in cell size heterogeneity ( Figure S3 ). Together, these data suggest that LL-37 and its derivatives bind and actively stimulate PhoQ. In the absence of MgrB, these peptides further enhance PhoQ activation. Importantly, cell elongation serves as a sensitive phenotype for peptide-mediated PhoQ stimulation in E. coli . Wild-type cells exposed to sub-MIC peptide concentrations exhibit moderate elongation, reflecting intermediate PhoQ activity, while ΔmgrB cells display significantly increased cell lengths, indicating robust PhoQ activation ( Figure 4E ). Discussion Despite extensive investigation, fundamental questions remain about the molecular mechanisms of peptide-mediated signal transduction by the PhoQ-PhoP system. Moreover, the effects of PhoPQ activation by cationic AMPs in cells and the physiological importance of these interactions have not been elucidated. We address two critical questions in this work: (i) Can peptides lacking potent antimicrobial activity still interact with PhoQ and act as a signal for this pathway? Using LL-37 and derivatives with variable MICs and antibacterial activities against E. coli cells, we show that the signaling function of peptides through the PhoQ-PhoP system is separable from their antimicrobial activity ( Figure 5A ), suggesting that bacteria may use this pathway primarily as a detection mechanism for host environments rather than simply as a response to membrane damage. (ii) Do the peptides actively stimulate PhoQ, or do they exert their effect on PhoQ passively by displacing the inhibitor protein, MgrB? Many studies on peptide-induced signaling of PhoQ measure the activity of peptides using PhoP-dependent transcriptional reporters as a proxy. In agreement with the previous studies, upon exposure to peptides, we do not observe a significant increase in PhoQP-driven transcriptional reporter activity in ΔmgrB cells relative to the wild type. It is possible that the derepression of PhoP-regulated gene expression in the absence of MgrB could lead to lower sensitivity of the reporter signal, masking small increments in the amplitude. While such reporters are incredibly useful in determining the activation status of this pathway and measuring the transcription levels, we have a second, sensitive readout – a cell elongation or filamentation phenotype in response to peptide stress in E. coli . Based on our microscopy and cell length measurements, we find that cells elongate significantly upon exposure to sublethal concentrations of peptides, LL-37 and KR-12, even though the promoter reporters do not show a significant change in their activities. The extent of cell elongation may differ in response to specific peptides, as we note that filamentation induced by an AMP, C18G, in wild-type E. coli cells is quite robust, with an average cell length of > 20 μm ( 2 ). Upon deletion of MgrB, the average cell lengths increased further, and even RI-10-treated cells displayed cell elongation; this observation suggests that AMPs may activate PhoQ directly in addition to causing MgrB dissociation as proposed previously ( 23 ). Our findings elucidate PhoQ-dependent effects of these peptides and support a model where peptides play an active and direct role in PhoQ activation ( Figure 5B ). Download figure Open in new tab Figure 5. Schematic overview of peptide stress response through the E. coli PhoQ-PhoP signaling system. (A) At high concentrations, antimicrobial peptides affect membrane integrity and cause damage, leading to cell death. At sub-MIC levels, peptides are sensed by the signaling systems, such as PhoQ/PhoP, which control the gene expression under peptide stress, leading to physiological changes, including cell elongation and modification of the outer cell membrane. (B) At physiological magnesium concentration, PhoQ is strongly stimulated by specific cationic peptides, and some PhoQ molecules are inhibited by the small protein regulator, MgrB, leading to an intermediate level of PhoQ activation. In the absence of MgrB, PhoQ is not inhibited, and when cells are exposed to peptides, PhoQ is strongly activated, resulting in maximal activation of this signaling system. Our understanding of what characteristics of AMPs are required to activate the PhoP-PhoQ system is still evolving. A previous report found a correlation between the extent of positive charge, hydrophobicity, and amphipathicity of an AMP and its ability to activate PhoQ-PhoP ( 8 ). Intriguingly, the authors discovered that even a peptide with minimal positive charge (+1) but containing histidines was highly potent at activating the system, suggesting the existence of an activation mechanism more complex than thought. A later study with 117 AMPs from the Antimicrobial Peptide Database ( 30 ) showed that PhoQ is activated by a diverse set of AMPs, including the human cathelicin LL-37 and non-cathelicidin peptides that carry either an α-helical or a combination of α-helical/β-sheet structural regions ( 31 ). In our work, we see that LL-37 derivatives induce PhoQ-dependent morphological changes, and KR-12, the smallest antibacterial peptide, is also the smallest peptide sequence for the PhoPQ induction. Further, RI-10, but not KE-10, became responsive upon the deletion of mgrB . Interestingly, LL-25, the N-terminal segment of LL-37 which does not kill bacteria under normal conditions ( 31 ), does not activate PhoPQ based on the recent human AMPs screen study although it contains partial sequence of KR-12 ( 30 ). LL-25 can be obtained by extending the sequence of KE-10 from both ends ( Tables S2 , S3 ). Together, the data from the current study suggest that part of the LL-37 amino acid sequence that overlaps with both KR-12 and RI-10 is likely mediating the interaction with PhoQ. More broadly, our findings imply that the bacterial molecular detection machinery is elegantly tuned to specifically target the core antimicrobial region of human antimicrobial peptide LL-37 but not fragments outside this region. When broadly viewed from the host-pathogen interaction at the molecular level, bacterial detection of the core antimicrobial region of human cathelicidin LL-37 ( 24 ) is remarkably efficient since this region is commonly shared by all the active fragments of LL-37 naturally produced in human skin ( Table S3 ), including KS-27, KR-20, KS-30, RK-31, and LL-29 ( 32 ). Indeed, all these human cathelicidin fragments responded to the PhoPQ system based on a recent screening of human AMPs registered in the APD ( 30 ). Collectively, these findings underscore that the bacterial PhoQ-PhoP system likely senses host defense peptides based on evolutionarily conserved structural and chemical properties independent of their antimicrobial activity, allowing bacteria to mount preemptive responses rather than simply react to membrane damage. Finally, the dramatic rise of antimicrobial resistance is a major public health concern. AMPs and other peptide derivatives are relatively small, typically comprised of around 10 to 50 amino acids ( 30 ), and constitute a key component of host innate immunity for defense against microbial pathogens ( 32 , 33 ). These cationic peptides are relatively cheap to produce, have good thermal stability and water solubility, and exhibit a broad spectrum of activity against bacteria and fungi, making them promising candidates for drug development ( 34 – 38 ). Studying the bacterial responses to antimicrobial peptides in general provides us with a fundamental understanding of how pathogens can sense, adapt, and develop tolerance to these host defense molecules and guide the strategic design of novel therapeutics with enhanced efficacy to overcome the growing antibiotic resistance problem. Materials and Methods Strains Details of the strains used in this study are provided in Table S4 . AML20, JNC21, NRS5, NRS6, SAM31, TIM92, TIM100, and TIM166 are E. coli K-12 MG1655 derivatives. Additionally, clinical strains of E. coli , E423-17, E416-17, and ATCC strain 25922 were used for specific assays as indicated. NRS6 was made by transducing the KEIO strain JW1815 into the TIM148 and TIM166 strains, respectively, using P1 vir . Media, reagents, and growth conditions Routine bacterial growth on solid agar was performed using LB Miller medium (IBI Scientific) containing 1.5% bacteriological grade agar (VWR Lifesciences) at 37°C. Liquid cultures were grown at 37°C with aeration in either LB miller medium or minimal A medium (MinA) ( 39 ) (K 2 HPO 4 (10.5 g l -1 ), KH 2 PO 4 (4.5 g l -1 ), (NH 4 ) 2 SO 4 (1.0 g l -1 ), Na 3 citrate.2H 2 O (0.5 g l -1 ), supplemented with 0.1% casamino acids, 0.2% glucose and 1 mM MgSO 4 unless otherwise indicated. Throughout this study, supplemented MinA, as indicated above, will be referred to as a minimal medium. In addition, Mueller-Hinton broth (MHB, catalog info) and tryptic soy broth (TSB, catalog info) were used for minimum inhibitory/bactericidal concentration (MIC/MBC) assays. Peptides and/or AMPs used in the study are indicated in Table S2 . For the measurement of cell length and transcriptional reporter activity, overnight cultures of SAM31, TIM92, TIM100, AML20, SAA58, and JNC21 were grown in supplemented MinA containing 1 mM MgSO 4 . Saturated cultures were back diluted 1:500 into 5 ml supplemented MinA with 0.1 mM Mg 2+ and grown for 2 hours in a shaker at 300 rpm. Each culture was split into two tubes, with one tube designated as a no-peptide control and the second tube where a test peptide was added. LL-37, KR-12, RI-10, and KE-10 were added to a final concentration of 1.25 µM, 5 µM, 5 µM, and 5 µM, respectively. These cultures were further grown for 4 hours at 37°C in a roller drum at 60 rpm. Microscopy and image analysis Cell morphology was observed and single-cell fluorescence to measure transcriptional reporter activity was performed using phase contrast and fluorescence microscopy as previously described ( 2 ). 3 µl of the cells were immobilized on a 1% agarose pad and observed using a Nikon TiE fluorescent microscope with a TI2-S-HU attachable mechanical stage and a perfect focus system (PFS). The images were captured using a Teledyne Photometrics Prime 95B sCMOS camera with 1×1 binning. YFP and CFP fluorescence was imaged using a 100 ms exposure time at 20% intensity. Image acquisition was done using Metamorph software version 7.10.3.279 (Molecular Devices, Sunnyvale, CA). The background fluorescence was determined using MG1655 cells grown under the same conditions. The average cell fluorescence was quantified using ImageJ ( 40 ) and the MicrobeJ plugin ( 41 ). Data from independent replicates were plotted using R. Minimum inhibitory/bactericidal concentration (MIC/MBC) testing The antibacterial activity of the peptides against E. coli MG1655, E423-17, E416-17, and 25922 strains was evaluated using a previously established protocol ( 42 ). Briefly, a peptide concentration gradient with twofold dilution was prepared in the 96-well polystyrene microplate at 10 μl per well. Bacteria were grown to the exponential phase in MHB, TSB, or supplemented MinA. It was then diluted to ∼10 5 CFU/ml in MHB, TSB, or supplemented MinA with 0.1 mM MgSO 4 , and partitioned into the 96-well microplate at 90 μl per well. After overnight incubation at 37°C, readings were taken at 600 nm using a ChroMate 4300 Microplate Reader at (GMI, Ramsey, MN). The minimum inhibitory concentration (MIC), represented as ranges, was determined for wells without bacterial growth. For minimum bactericidal concentration (MBC) determination, aliquots of 30 μl from growth-absent wells were plated on MH agar plates, and the lowest peptide concentration with no isolated colony was considered the MBC. Results were expressed in µM. Membrane permeabilization The peptide ladder was prepared as described for the antibacterial assay in a black COSTAR 96-well plate. Propidium iodide (PI) (MP Biomedicals, Solon, OH) was prepared in the dark and dissolved in DMSO (Thermo Fisher Scientific, NY) to 20 mM. This PI stock solution was further diluted to 1 mM with water and 2 μl of 1 mM PI was added to each well. An overnight culture of E. coli K12 MG1655 was inoculated into 1xMinA supplemented with 0.2% glucose, 0.1% casamino acids, 0.1mM MgSO 4 , and grown to exponential phase. It was then diluted to OD 600 0.11 in the same media, and 88 μl was added to each well. The plate was incubated at 37℃ with continuous shaking in a FLUOstar Omega (BMG LABTECH, NC) microplate reader. The sample was read every 5 minutes for 24 cycles with excitation and emission wavelengths of 584 nm and 620 nm, respectively. Killing kinetics E. coli K12 MG1655 was grown to the exponential phase (OD 600 ∼ 0.3) in 1xMinA supplemented with 0.2% glucose, 0.1% casamino acids, 1 mM MgSO 4 . The culture was diluted to OD 600 0.001 (∼10 5 CFU/ml) in 1xMinA supplemented with 0.2% glucose, 0.1% casamino acids, 0.1mM MgSO 4 . One milliliter of bacterial suspension was then mixed with LL-37 (4 μM, 2× MIC), KR-12 (8 μM, 2× MIC) or RI-10 (32 μM) and incubated at 37°C. After 15, 30, 60, 90, and 120 minutes of incubation, 50 μl was taken and serially diluted with 1×PBS. Then, 50 μl of the diluted suspension was plated on MH agar Agar plates. The plates were incubated overnight at 37°C for bacterial CFU determination. Membrane depolarization An overnight culture of E. coli K12 MG1655 was inoculated into 1xMinA supplemented with 0.2% glucose, 0.1% casamino acids, 1 mM MgSO 4 , and grown to exponential phase. Bacteria were washed with 1×PBS, re-suspended in twice the volume of 1×PBS containing 25 mM glucose, and incubated at 37°C for 15 minutes. Then, 500 nM (final concentration) of the DiBAC4 ( 3 ) bis-(1,3-dibutylbarbituric acid) trimethine oxonol (ANASPEC, CA) was added and vortexed gently. Aliquots of 90 µl of the energized bacteria solution were loaded to the 96 well plates (Corning COSTAR, AZ) and placed in a FLUOstar Omega microplate reader (BMG LABTECH, NC). Fluorescence was read for 15 min at excitation and emission wavelengths of 485 nm and 520 nm, respectively. Then, 10 µl of peptide solutions were added and fluorescence readings were recorded for 45 min. Triton X-100 (0.1%) was used as a positive control. Funding S.S.Y. is supported by the National Institutes of Health - National Institute of General Medical Sciences (NIH-NIGMS) ESI-MIRA R35 GM147566 and institutional start-up funds from Rutgers. A.M. is supported by the Waksman Institute Busch Predoctoral fellowship (2024-25) at Rutgers. G.W. is supported by the NIH/NIAID R56 AI175209. The funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author contributions S.S.Y. and G.W. conceived the experiments. All authors planned the experiments, designed the methodology, and performed data analysis. S.A.A., A.F.M., and A.M. performed the experiments. All authors contributed to the writing and editing of the manuscript. Competing interests The authors declare no competing financial interests. Materials and resource availability Requests for strains and plasmids generated in this study should be directed to and will be fulfilled by the lead contact, Srujana S. Yadavalli. Further information and requests for resources and reagents should be directed to and will be fulfilled by Srujana S. Yadavalli and Guangshun Wang. Data availability The main manuscript and its supporting files include all data generated or analyzed during this study. Supporting Information Tables View this table: View inline View popup Download powerpoint Table S1. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values for LL-37 and its derivatives against E. coli strains. MIC values (µM) of antimicrobial peptide LL-37 and its derivatives KR-12 and RI-10 against different E. coli strains in supplemented minimal medium (Min A), Mueller Hinton broth (MHB), and tryptic soy broth (TSB). The corresponding MBC values (µM) are shown within parentheses. Data represent values obtained from three independent biological replicates. View this table: View inline View popup Download powerpoint Table S2. List of peptides used in the study. View this table: View inline View popup Download powerpoint Table S3. List of human LL-37 fragments that share the key recognition sequence for bacterial sensing (bolded). View this table: View inline View popup Download powerpoint Table S4. List of strains used in the study. Figures Download figure Open in new tab Figure S1. Killing kinetics LL-37, KR-12, and RI-10 against E. coli K-12 MG1655. Cells were treated with peptides at indicated concentrations in supplemented MinA with 0.1 mM MgSO 4 . Data are represented as mean ± range for two biological replicates. Download figure Open in new tab Figure S2. PhoQP-regulated transcription and cell lengths in mgrB-t7a reporter cells treated with sub-MIC levels of antimicrobial peptides. (A, C) PhoQP-dependent promoter reporter activity in wild-type (TIM166) or ΔmgrB (NRS6) cells, and (B, D) quantification of cell lengths of wild-type or ΔmgrB cells, grown in the presence of sub-MIC levels of the antimicrobial peptides LL-37 (1.25 µM), KR-12 (5 µM), RI-10 (5 µM), and KE-10 (5 µM) in supplemented MinA with 0.1 mM MgSO 4 . Data are obtained from three biological replicates; mean and median values are shown in red text and black bars, respectively. The number of cells analyzed is indicated for each sample. P-values indicate the results of a Student’s t-test: ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05, and “ns” = P > 0.05. Download figure Open in new tab Figure S3. PhoQP-regulated transcription and cell lengths in ΔmgrB ΔqueE cells treated with sub-MIC levels of antimicrobial peptides. (A) PhoQP-dependent promoter reporter activity, (B) quantification of cell lengths, and (C) phase contrast micrographs of ΔmgrB ΔqueE (JNC21) cells grown in the presence of sub-MIC levels of the antimicrobial peptides LL-37 (1.25 µM), KR-12 (5 µM), RI-10 (5 µM), and KE-10 (5 µM) in supplemented MinA with 0.1 mM MgSO 4 . Data are obtained from three biological replicates; mean and median values are shown in red text and black bars, respectively. The number of cells analyzed is indicated for each sample. P-values indicate the results of a Student’s t-test: ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05, and “ns” = P > 0.05. Acknowledgments The authors thank Dr. Mark Goulian for sharing transcriptional reporter strains for mgrB , Dr. Nitish Sharma for preparing ΔmgrB strains carrying the reporters, Dr. Premal Shah for critical feedback and assistance with data visualization and figure creation, Dr. Manuela Roggiani for critical feedback on the manuscript, and members of the Yadavalli and Wang labs for helpful discussions. Funder Information Declared National Institute of General Medical Sciences, https://ror.org/04q48ey07 , R35 GM147566 National Institute of Allergy and Infectious Diseases, https://ror.org/043z4tv69 , R56 AI175209 Footnotes Figures 2, 3, 4, S2, and S3 (specifically the graphical plots in the figures). Minor edits to the abstract, introduction, results, discussion, and figure legends in the main manuscript + SI. References 1. ↵ M. W. Bader , et al. , Recognition of antimicrobial peptides by a bacterial sensor kinase . Cell 122 , 461 – 472 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 2. ↵ S. S. Yadavalli , et al. , Antimicrobial peptides trigger a division block in Escherichia coli through stimulation of a signalling system . Nat. Commun . 7 , 12340 ( 2016 ). OpenUrl CrossRef PubMed 3. ↵ E. A. Groisman , The pleiotropic two-component regulatory system PhoP-PhoQ . J. Bacteriol . 183 , 1835 – 1842 ( 2001 ). OpenUrl FREE Full Text 4. ↵ J. Yuan , F. Jin , T. Glatter , V. Sourjik , Osmosensing by the bacterial PhoQ/PhoP two-component system . Proc. Natl. Acad. Sci. U. S. A . 114 , E10792 – E10798 ( 2017 ). OpenUrl Abstract / FREE Full Text 5. ↵ S. Matamouros , S. I. Miller , S. Typhimurium strategies to resist killing by cationic antimicrobial peptides . Biochim. Biophys. Acta 1848 , 3021 – 3025 ( 2015 ). OpenUrl CrossRef 6. Z. D. Dalebroux , S. I. Miller , Salmonellae PhoPQ regulation of the outer membrane to resist innate immunity . Curr. Opin. Microbiol . 17 , 106 – 113 ( 2014 ). OpenUrl CrossRef PubMed 7. R. K. Ernst , T. Guina , S. I. Miller , Salmonella typhimurium outer membrane remodeling: role in resistance to host innate immunity . Microbes Infect . 3 , 1327 – 1334 ( 2001 ). OpenUrl CrossRef PubMed Web of Science 8. ↵ T. Shprung , A. Peleg , Y. Rosenfeld , P. Trieu-Cuot , Y. Shai , Effect of PhoP-PhoQ activation by broad repertoire of antimicrobial peptides on bacterial resistance . J. Biol. Chem . 287 , 4544 – 4551 ( 2012 ). OpenUrl Abstract / FREE Full Text 9. ↵ M. F. Burton , P. G. Steel , The chemistry and biology of LL-37 . Nat. Prod. Rep . 26 , 1572 – 1584 ( 2009 ). OpenUrl CrossRef PubMed 10. D. Vandamme , B. Landuyt , W. Luyten , L. Schoofs , A comprehensive summary of LL-37, the factotum human cathelicidin peptide . Cell. Immunol . 280 , 22 – 35 ( 2012 ). OpenUrl CrossRef PubMed 11. ↵ G. Wang , et al. , Design of antimicrobial peptides: Progress made with human cathelicidin LL-37 . Adv. Exp. Med. Biol . 1117 , 215 – 240 ( 2019 ). OpenUrl PubMed 12. ↵ F. Lu , Y. Zhu , G. Zhang , Z. Liu , Renovation as innovation: Repurposing human antibacterial peptide LL-37 for cancer therapy . Front. Pharmacol . 13 , 944147 ( 2022 ). OpenUrl PubMed 13. K. Niemirowicz , et al. , Magnetic nanoparticles enhance the anticancer activity of cathelicidin LL-37 peptide against colon cancer cells . Int. J. Nanomedicine 10 , 3843 – 3853 ( 2015 ). OpenUrl PubMed 14. K. Kuroda , K. Okumura , H. Isogai , E. Isogai , The human cathelicidin antimicrobial peptide LL-37 and mimics are potential anticancer drugs . Front. Oncol . 5 , 144 ( 2015 ). OpenUrl PubMed 15. ↵ S. Tripathi , et al. , Antiviral activity of the human cathelicidin, LL-37, and derived peptides on seasonal and pandemic influenza A viruses . PLoS One 10 , e0124706 ( 2015 ). OpenUrl CrossRef PubMed 16. ↵ I. Ginsburg , Bactericidal cationic peptides can also function as bacteriolysis-inducing agents mimicking beta-lactam antibiotics?; it is enigmatic why this concept is consistently disregarded . Med. Hypotheses 62 , 367 – 374 ( 2004 ). OpenUrl CrossRef PubMed 17. Y. Zhang , J. Lakshmaiah Narayana , Q. Wu , X. Dang , G. Wang , Structure and activity of a selective antibiofilm peptide SK-24 derived from the NMR structure of human cathelicidin LL-37 . Pharmaceuticals (Basel ) 14 , 1245 ( 2021 ). OpenUrl PubMed 18. ↵ K. A. Henzler Wildman , D.-K. Lee , A. Ramamoorthy , Mechanism of lipid bilayer disruption by the human antimicrobial peptide, LL-37 . Biochemistry 42 , 6545 – 6558 ( 2003 ). OpenUrl CrossRef PubMed Web of Science 19. ↵ J. Turner , Y. Cho , N. N. Dinh , A. J. Waring , R. I. Lehrer , Activities of LL-37, a cathelin-associated antimicrobial peptide of human neutrophils . Antimicrob. Agents Chemother . 42 , 2206 – 2214 ( 1998 ). OpenUrl Abstract / FREE Full Text 20. ↵ A. M. Lippa , M. Goulian , Feedback inhibition in the PhoQ/PhoP signaling system by a membrane peptide . PLoS Genet . 5 , e1000788 ( 2009 ). OpenUrl CrossRef PubMed 21. ↵ M. E. Salazar , A. I. Podgornaia , M. T. Laub , The small membrane protein MgrB regulates PhoQ bifunctionality to control PhoP target gene expression dynamics . Mol. Microbiol . 102 , 430 – 445 ( 2016 ). OpenUrl CrossRef PubMed 22. ↵ S. S. Yadavalli , et al. , Functional determinants of a small protein controlling a broadly conserved bacterial sensor kinase . J. Bacteriol . 202 ( 2020 ). 23. ↵ S. Jiang , et al. , The inhibitory mechanism of a small protein reveals its role in antimicrobial peptide sensing . Proc. Natl. Acad. Sci. U. S. A . 120 , e2309607120 ( 2023 ). OpenUrl CrossRef PubMed 24. ↵ G. Wang , Structures of human host defense cathelicidin LL-37 and its smallest antimicrobial peptide KR-12 in lipid micelles . J. Biol. Chem . 283 , 32637 – 32643 ( 2008 ). OpenUrl Abstract / FREE Full Text 25. ↵ K. Chen , W. Gong , J. Huang , T. Yoshimura , J. M. Wang , The potentials of short fragments of human anti-microbial peptide LL-37 as a novel therapeutic modality for diseases . Front. Biosci. (Landmark Ed .) 26 , 1362 – 1372 ( 2021 ). OpenUrl CrossRef PubMed 26. ↵ J. Lakshmaiah Narayana , et al. , Origami of KR-12 designed antimicrobial peptides and their potential applications . Antibiotics (Basel ) 13 , 816 ( 2024 ). OpenUrl PubMed 27. ↵ N. Mookherjee , M. A. Anderson , H. P. Haagsman , D. J. Davidson , Antimicrobial host defence peptides: functions and clinical potential . Nat. Rev. Drug Discov . 19 , 311 – 332 ( 2020 ). OpenUrl CrossRef PubMed 28. ↵ S. A. Adeleye , S. S. Yadavalli , Queuosine biosynthetic enzyme, QueE moonlights as a cell division regulator . PLoS Genet . 20 , e1011287 ( 2024 ). OpenUrl CrossRef PubMed 29. ↵ T. Miyashiro , M. Goulian , Stimulus-dependent differential regulation in the Escherichia coli PhoQ PhoP system . Proc. Natl. Acad. Sci. U. S. A . 104 , 16305 – 16310 ( 2007 ). OpenUrl Abstract / FREE Full Text 30. ↵ G. Wang , X. Li , Z. Wang , APD6: the antimicrobial peptide database is expanded to promote research and development by deploying an unprecedented information pipeline . Nucleic Acids Res . in Press ( 2025 ). 31. ↵ K. R. Brink , et al. , An E. coli display method for characterization of peptide-sensor kinase interactions . Nat. Chem. Biol . 19 , 451 – 459 ( 2023 ). OpenUrl CrossRef PubMed 32. ↵ G. Wang , et al. , Structure, dynamics, and antimicrobial and immune modulatory activities of human LL-23 and its single-residue variants mutated on the basis of homologous primate cathelicidins . Biochemistry 51 , 653 – 664 ( 2012 ). OpenUrl CrossRef PubMed 33. ↵ M. Murakami , B. Lopez-Garcia , M. Braff , R. A. Dorschner , R. L. Gallo , Postsecretory processing generates multiple cathelicidins for enhanced topical antimicrobial defense . J. Immunol . 172 , 3070 – 3077 ( 2004 ). OpenUrl Abstract / FREE Full Text 34. ↵ G. Laverty , S. P. Gorman , B. F. Gilmore , The potential of antimicrobial peptides as biocides . Int. J. Mol. Sci . 12 , 6566 – 6596 ( 2011 ). OpenUrl CrossRef PubMed 35. L. Segev-Zarko , M. L. Mangoni , Y. Shai , “Antimicrobial peptides: multiple mechanisms against a variety of targets” in Antimicrobial Peptides: Discovery, Design and Novel Therapeutic Strategies , (CABI , 2017 ), pp. 119 – 134 . 36. R. E. W. Hancock , K. L. Brown , N. Mookherjee , Host defence peptides from invertebrates--emerging antimicrobial strategies . Immunobiology 211 , 315 – 322 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 37. T. L. Raguse , E. A. Porter , B. Weisblum , S. H. Gellman , Structure-activity studies of 14-helical antimicrobial beta-peptides: probing the relationship between conformational stability and antimicrobial potency . J. Am. Chem. Soc . 124 , 12774 – 12785 ( 2002 ). OpenUrl CrossRef PubMed 38. ↵ D. L. Lee , R. S. Hodges , Structure-activity relationships of de novo designed cyclic antimicrobial peptides based on gramicidin S . Biopolymers 71 , 28 – 48 ( 2003 ). OpenUrl CrossRef PubMed 39. ↵ J. H. Miller , A Short Course in Bacterial Genetics , 1987th Ed. ( Cold Spring Harbor Laboratory Press , 1992 ). 40. ↵ C. A. Schneider , W. S. Rasband , K. W. Eliceiri , NIH Image to ImageJ: 25 years of image analysis . Nat. Methods 9 , 671 – 675 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 41. ↵ A. Ducret , E. M. Quardokus , Y. V. Brun , MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis . Nat. Microbiol . 1 , 16077 ( 2016 ). 42. ↵ B. Mishra , T. Lushnikova , R. M. Golla , X. Wang , G. Wang , Design and surface immobilization of short anti-biofilm peptides . Acta Biomater . 49 , 316 – 328 ( 2017 ). OpenUrl CrossRef PubMed References 1. J. Turner , Y. Cho , N. N. Dinh , A. J. Waring , R. I. Lehrer , Activities of LL-37, a cathelin-associated antimicrobial peptide of human neutrophils . Antimicrob. Agents Chemother . 42 , 2206 – 2214 ( 1998 ). OpenUrl Abstract / FREE Full Text 2. G. Wang , Structures of human host defense cathelicidin LL-37 and its smallest antimicrobial peptide KR-12 in lipid micelles . J. Biol. Chem . 283 , 32637 – 32643 ( 2008 ). OpenUrl Abstract / FREE Full Text 3. R. F. Epand , G. Wang , B. Berno , R. M. Epand , Lipid segregation explains selective toxicity of a series of fragments derived from the human cathelicidin LL-37 . Antimicrob. Agents Chemother . 53 , 3705 – 3714 ( 2009 ). OpenUrl Abstract / FREE Full Text 4. A. F. Mechesso , Y. Su , J. Xie , G. Wang , Enhanced antimicrobial screening sensitivity enabled the identification of an ultrashort peptide KR-8 for engineering of LL-37mini to combat drug-resistant pathogens . ACS Infect. Dis . 9 , 2215 – 2225 ( 2023 ). OpenUrl PubMed 5. A. M. Lippa , M. Goulian , Feedback inhibition in the PhoQ/PhoP signaling system by a membrane peptide . PLoS Genet . 5 , e1000788 ( 2009 ). OpenUrl CrossRef PubMed 6. S. S. Yadavalli , et al. , Antimicrobial peptides trigger a division block in Escherichia coli through stimulation of a signalling system . Nat. Commun . 7 , 12340 ( 2016 ). OpenUrl CrossRef PubMed 7. T. Miyashiro , M. Goulian , Stimulus-dependent differential regulation in the Escherichia coli PhoQ PhoP system . Proc. Natl. Acad. Sci. U. S. A . 104 , 16305 – 16310 ( 2007 ). OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted October 23, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Mechanistic Insights into Human Antimicrobial Peptide-Induced Activation of a Broadly Conserved Bacterial Signaling System Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Mechanistic Insights into Human Antimicrobial Peptide-Induced Activation of a Broadly Conserved Bacterial Signaling System Samuel A. Adeleye , Abraham F. Mechesso , Arpita Mukherjee , Guangshun Wang , Srujana S. Yadavalli bioRxiv 2025.05.06.652532; doi: https://doi.org/10.1101/2025.05.06.652532 Share This Article: Copy Citation Tools Mechanistic Insights into Human Antimicrobial Peptide-Induced Activation of a Broadly Conserved Bacterial Signaling System Samuel A. Adeleye , Abraham F. Mechesso , Arpita Mukherjee , Guangshun Wang , Srujana S. Yadavalli bioRxiv 2025.05.06.652532; doi: https://doi.org/10.1101/2025.05.06.652532 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 Microbiology Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42005) Biophysics (21472) Cancer Biology (18624) Cell Biology (25534) Clinical Trials (138) Developmental Biology (13391) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40437) Molecular Biology (17200) Neuroscience (88697) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4829) Physiology (7653) Plant Biology (15171) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9827) Zoology (2272)