Identification of a Genetic Locus Responsible for Antimicrobial Peptide Resistance inClostridium difficile

ABSTRACTClostridium difficilecauses chronic intestinal disease, yet little is understood about how the bacterium interacts with and survives in the host. To colonize the intestine and cause persistent disease, the bacterium must circumvent killing by host innate immune factors, such as cationic antimicrobial peptides (CAMPs). In this study, we investigated the effect of model CAMPs on growth and found thatC. difficileis not only sensitive to these compounds but also responds to low levels of CAMPs by expressing genes that lead to CAMP resistance. By plating the bacterium on medium containing the CAMP nisin, we isolated a mutant capable of growing in three times the inhibitory concentration of CAMPs. This mutant also showed increased resistance to the CAMPs gallidermin and polymyxin B, demonstrating tolerance to different types of antimicrobial peptides. We identified the mutated gene responsible for the resistance phenotype as CD1352. This gene encodes a putative orphan histidine kinase that lies adjacent to a predicted ABC transporter operon (CD1349 to CD1351). Transcriptional analysis of the ABC transporter genes revealed that this operon was upregulated in the presence of nisin in wild-type cells and was more highly expressed in the CD1352 mutant. The insertional disruption of the CD1349 gene resulted in significant decreases in resistance to the CAMPs nisin and gallidermin but not polymyxin B. Because of their role in cationic antimicrobial peptide resistance, we propose the designationcprABCfor genes CD1349 to CD1351 andcprKfor the CD1352 gene. These results provide the first evidence of aC. difficilegene associated with antimicrobial peptide resistance.

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ABC Transporter DerAB of Lactobacillus casei Mediates Resistance against Insect-Derived Defensins

Applied and Environmental Microbiology ◽

10.1128/aem.00818-20 ◽

2020 ◽

Vol 86 (14) ◽

Author(s):

Ainhoa Revilla-Guarinos ◽

Qian Zhang ◽

Christoph Loderer ◽

Cristina Alcántara ◽

Ariane Müller ◽

...

Keyword(s):

Antimicrobial Peptides ◽

Antimicrobial Peptide ◽

Abc Transporter ◽

Lactobacillus Casei ◽

Biological Effects ◽

Gc Content ◽

Resistance Response ◽

Content Type ◽

Antimicrobial Peptide Resistance ◽

High Level

ABSTRACT Bce-like systems mediate resistance against antimicrobial peptides in Firmicutes bacteria. Lactobacillus casei BL23 encodes an “orphan” ABC transporter that, based on homology to BceAB-like systems, was proposed to contribute to antimicrobial peptide resistance. A mutant lacking the permease subunit was tested for sensitivity against a collection of peptides derived from bacteria, fungi, insects, and humans. Our results show that the transporter specifically conferred resistance against insect-derived cysteine-stabilized αβ defensins, and it was therefore renamed DerAB for defensin resistance ABC transporter. Surprisingly, cells lacking DerAB showed a marked increase in resistance against the lantibiotic nisin. This could be explained by significantly increased expression of the antimicrobial peptide resistance determinants regulated by the Bce-like systems PsdRSAB (formerly module 09) and ApsRSAB (formerly module 12). Bacterial two-hybrid studies in Escherichia coli showed that DerB could interact with proteins of the sensory complex in the Psd resistance system. We therefore propose that interaction of DerAB with this complex in the cell creates signaling interference and reduces the cell’s potential to mount an effective nisin resistance response. In the absence of DerB, this negative interference is relieved, leading to the observed hyperactivation of the Psd module and thus increased resistance to nisin. Our results unravel the function of a previously uncharacterized Bce-like orphan resistance transporter with pleiotropic biological effects on the cell. IMPORTANCE Antimicrobial peptides (AMPs) play an important role in suppressing the growth of microorganisms. They can be produced by bacteria themselves—to inhibit competitors—but are also widely distributed in higher eukaryotes, including insects and mammals, where they form an important component of innate immunity. In low-GC-content Gram-positive bacteria, BceAB-like transporters play a crucial role in AMP resistance but have so far been primarily associated with interbacterial competition. Here, we show that the orphan transporter DerAB from the lactic acid bacterium Lactobacillus casei is crucial for high-level resistance against insect-derived AMPs. It therefore represents an important mechanism for interkingdom defense. Furthermore, our results support a signaling interference from DerAB on the PsdRSAB module that might prevent the activation of a full nisin response. The Bce modules from L. casei BL23 illustrate a biological paradox in which the intrinsic nisin detoxification potential only arises in the absence of a defensin-specific ABC transporter.

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Antimicrobial Peptides: A Promising Avenue for Human Healthcare

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666191011121722 ◽

2020 ◽

Vol 21 (2) ◽

pp. 90-96 ◽

Cited By ~ 2

Author(s):

Girish M. Bhopale

Keyword(s):

Antimicrobial Peptides ◽

Antimicrobial Peptide ◽

Antimicrobial Agents ◽

Current Status ◽

Antimicrobial Drugs ◽

Spectrum Activity ◽

Low Levels ◽

Human Healthcare ◽

Broad Spectrum Activity ◽

Promising Avenue

Antimicrobial drugs resistant microbes have been observed worldwide and therefore alternative development of antimicrobial peptides has gained interest in human healthcare. Enormous progress has been made in the development of antimicrobial peptide during the last decade due to major advantages of AMPs such as broad-spectrum activity and low levels of induced resistance over the current antimicrobial agents. This review briefly provides various categories of AMP, their physicochemical properties and mechanism of action which governs their penetration into microbial cell. Further, the recent information on current status of antimicrobial peptide development, their applications and perspective in human healthcare are also described.

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Polymyxin B Resistance in El Tor Vibrio cholerae Requires Lipid Acylation Catalyzed by MsbB

Journal of Bacteriology ◽

10.1128/jb.00023-10 ◽

2010 ◽

Vol 192 (8) ◽

pp. 2044-2052 ◽

Cited By ~ 31

Author(s):

Jyl S. Matson ◽

Hyun Ju Yoo ◽

Kristina Hakansson ◽

Victor J. DiRita

Keyword(s):

Antimicrobial Peptides ◽

Vibrio Cholerae ◽

Lipid A ◽

Polymyxin B ◽

Mass Spectrometry Analysis ◽

Peptide Antibiotic ◽

Wild Type ◽

Spectrometry Analysis ◽

Antimicrobial Peptide Resistance ◽

El Tor

ABSTRACTAntimicrobial peptides are critical for innate antibacterial defense. Both Gram-negative and Gram-positive microbes have mechanisms to alter their surfaces and resist killing by antimicrobial peptides. InVibrio cholerae, two natural epidemic biotypes, classical and El Tor, exhibit distinct phenotypes with respect to sensitivity to the peptide antibiotic polymyxin B: classical strains are sensitive and El Tor strains are relatively resistant. We carried out mutant screens of both biotypes, aiming to identify classicalV. choleraemutants resistant to polymyxin B and El TorV. choleraemutants sensitive to polymyxin B. Insertions in a gene annotatedmsbB(encoding a predicted lipid A secondary acyltransferase) answered both screens, implicating its activity in antimicrobial peptide resistance ofV. cholerae. Analysis of a defined mutation in the El Tor biotype demonstrated thatmsbBis required for resistance to all antimicrobial peptides tested. Mutation ofmsbBin a classical strain resulted in reduced resistance to several antimicrobial peptides but in no significant change in resistance to polymyxin B.msbBmutants of both biotypes showed decreased colonization of infant mice, with a more pronounced defect observed for the El Tor mutant. Mass spectrometry analysis showed that lipid A of themsbBmutant for both biotypes was underacylated compared to lipid A of the wild-type isolates, confirming that MsbB is a functional acyltransferase inV. cholerae.

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An iron-regulated LysR-type element mediates antimicrobial peptide resistance and virulence in Yersinia pseudotuberculosis

Microbiology ◽

10.1099/mic.0.026690-0 ◽

2009 ◽

Vol 155 (7) ◽

pp. 2168-2181 ◽

Cited By ~ 10

Author(s):

Sonia Arafah ◽

Marie-Laure Rosso ◽

Linda Rehaume ◽

Robert E. W. Hancock ◽

Michel Simonet ◽

...

Keyword(s):

Antimicrobial Peptide ◽

Yersinia Pseudotuberculosis ◽

Polymyxin B ◽

Uptake System ◽

Mesenteric Lymph Nodes ◽

Iron Starvation ◽

Iron Deprivation ◽

Genes Encoding ◽

Antimicrobial Peptide Resistance

During the course of its infection of the mammalian digestive tract, the entero-invasive, Gram-negative bacterium Yersinia pseudotuberculosis must overcome various hostile living conditions (notably, iron starvation and the presence of antimicrobial compounds produced in situ). We have previously reported that in vitro bacterial growth during iron deprivation raises resistance to the antimicrobial peptide polymyxin B; here, we show that this phenotype is mediated by a chromosomal gene (YPTB0333) encoding a transcriptional regulator from the LysR family. We determined that the product of YPTB0333 is a pleiotropic regulator which controls (in addition to its own expression) genes encoding the Yfe iron-uptake system and polymyxin B resistance. Lastly, by using a mouse model of oral infection, we demonstrated that YPTB0333 is required for colonization of Peyer's patches and mesenteric lymph nodes by Y. pseudotuberculosis.

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Induction of the Cpx Envelope Stress Pathway Contributes to Escherichia coli Tolerance to Antimicrobial Peptides

Applied and Environmental Microbiology ◽

10.1128/aem.02593-13 ◽

2013 ◽

Vol 79 (24) ◽

pp. 7770-7779 ◽

Cited By ~ 21

Author(s):

Bianca Audrain ◽

Lionel Ferrières ◽

Amira Zairi ◽

Guillaume Soubigou ◽

Curtis Dobson ◽

...

Keyword(s):

Escherichia Coli ◽

Antimicrobial Peptides ◽

Antimicrobial Peptide ◽

Polymyxin B ◽

Genetic Response ◽

Content Type ◽

E Coli ◽

Multiresistant Bacteria ◽

Envelope Stress ◽

Stress Pathway

ABSTRACTAntimicrobial peptides produced by multicellular organisms as part of their innate system of defense against microorganisms are currently considered potential alternatives to conventional antibiotics in case of infection by multiresistant bacteria. However, while the mode of action of antimicrobial peptides is relatively well described, resistance mechanisms potentially induced or selected by these peptides are still poorly understood. In this work, we studied the mechanisms of action and resistance potentially induced by ApoEdpL-W, a new antimicrobial peptide derived from human apolipoprotein E. Investigation of the genetic response ofEscherichia coliupon exposure to sublethal concentrations of ApoEdpL-W revealed that this antimicrobial peptide triggers activation of RcsCDB, CpxAR, and σEenvelope stress pathways. This genetic response is not restricted to ApoEdpL-W, since several other antimicrobial peptides, including polymyxin B, melittin, LL-37, and modified S4dermaseptin, also activate severalE. colienvelope stress pathways. Finally, we demonstrate that induction of the CpxAR two-component system directly contributes toE. colitolerance toward ApoEdpL-W, polymyxin B, and melittin. These results therefore show thatE. colisenses and responds to different antimicrobial peptides by activation of the CpxAR pathway. While this study further extends the understanding of the array of peptide-induced stress signaling systems, it also provides insight into the contribution of Cpx envelope stress pathway toE. colitolerance to antimicrobial peptides.

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SSD1 Is Integral to Host Defense Peptide Resistance in Candida albicans

Eukaryotic Cell ◽

10.1128/ec.00402-07 ◽

2008 ◽

Vol 7 (8) ◽

pp. 1318-1327 ◽

Cited By ~ 29

Author(s):

Kimberly D. Gank ◽

Michael R. Yeaman ◽

Satoshi Kojima ◽

Nannette Y. Yount ◽

Hyunsook Park ◽

...

Keyword(s):

Candida Albicans ◽

Antimicrobial Peptides ◽

Antimicrobial Peptide ◽

Host Defense ◽

Genomic Library ◽

Inflammatory Responses ◽

Host Defense Peptides ◽

Antimicrobial Peptide Resistance ◽

Null Mutants

ABSTRACT Candida albicans is usually a harmless human commensal. Because inflammatory responses are not normally induced by colonization, antimicrobial peptides are likely integral to first-line host defense against invasive candidiasis. Thus, C. albicans must have mechanisms to tolerate or circumvent molecular effectors of innate immunity and thereby colonize human tissues. Prior studies demonstrated that an antimicrobial peptide-resistant strain of C. albicans, 36082R, is hypervirulent in animal models versus its susceptible counterpart (36082S). The current study aimed to identify a genetic basis for antimicrobial peptide resistance in C. albicans. Screening of a C. albicans genomic library identified SSD1 as capable of conferring peptide resistance to a susceptible surrogate, Saccharomyces cerevisiae. Sequencing confirmed that the predicted translation products of 36082S and 36082R SSD1 genes were identical. However, Northern analyses corroborated that SSD1 is expressed at higher levels in 36082R than in 36082S. In isogenic backgrounds, ssd1Δ/ssd1Δ null mutants were significantly more susceptible to antimicrobial peptides than parental strains but had equivalent susceptibilities to nonpeptide stressors. Moreover, SSD1 complementation of ssd1Δ/ssd1Δ mutants restored parental antimicrobial peptide resistance phenotypes, and overexpression of SSD1 conferred enhanced peptide resistance. Consistent with these in vitro findings, ssd1 null mutants were significantly less virulent in a murine model of disseminated candidiasis than were their parental or complemented strains. Collectively, these results indicate that SSD1 is integral to C. albicans resistance to host defense peptides, a phenotype that appears to enhance the virulence of this organism in vivo.

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Phage-encoded cationic antimicrobial peptide used for outer membrane disruption in lysis

10.1101/515445 ◽

2019 ◽

Cited By ~ 2

Author(s):

Ashley Holt ◽

Jesse Cahill ◽

Jolene Ramsey ◽

Chandler O’Leary ◽

Russell Moreland ◽

...

Keyword(s):

Antimicrobial Peptides ◽

Antimicrobial Peptide ◽

Outer Membrane ◽

Genetic Selection ◽

Helical Structure ◽

Bioinformatic Analysis ◽

Membrane Disruption ◽

Bacterial Cells ◽

New Paradigm ◽

Cationic Antimicrobial Peptide

AbstractSpanins are required for the last step in bacteriophage lysis: the disruption of the outer membrane. Bioinformatic analysis has shown that ~15% of phages lack a spanin gene, which suggests an alternate mechanism of outer membrane disruption. To address this, we selected virulent podophage ϕKT as a spaninless exemplar and tested ϕKT genes for outer membrane disruption during lysis. Hypothetical novel gene 28 causes outer membrane disruption when co-expressed with ϕKT lysis genes and complements the lysis defect of a λ spanin mutant. Gp28 is a 56 aa cationic peptide with predicted amphipathic helical structure and is associated with the particulate fraction after lysis. Urea and KCl washes did not release gp28 from the particulate, suggesting a strong hydrophobic interaction with the membrane. Super high-resolution microscopy supports a primarily outer membrane localization for the peptide. Additionally, holin function is not required for gp28-mediated lysis. Gp28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. In standard assays to measure bactericidal and inhibitory effects of antimicrobial peptides on bacterial cells, synthesized gp28 performed equivalently to LL-37. The studies presented here suggest that ϕKT Gp28 disrupts bacterial outer membranes during lysis in a manner akin to antimicrobial peptides.SignificanceHere we provide evidence that ϕKT produces an antimicrobial peptide for outer membrane disruption during lysis. The disruptin is a new paradigm for phage lysis, and has no similarities to other known lysis genes. Many mechanisms have been proposed for the function of antimicrobial peptides, however there is not a consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic selection system to support such studies. Therefore, the ϕKT disruptin may represent the first genetically tractable antimicrobial peptide.

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A Protein Important for Antimicrobial Peptide Resistance, YdeI/OmdA, Is in the Periplasm and Interacts with OmpD/NmpC

Journal of Bacteriology ◽

10.1128/jb.00688-09 ◽

2009 ◽

Vol 191 (23) ◽

pp. 7243-7252 ◽

Cited By ~ 36

Author(s):

M. Carolina Pilonieta ◽

Kimberly D. Erickson ◽

Robert K. Ernst ◽

Corrella S. Detweiler

Keyword(s):

Gene Expression ◽

Antimicrobial Peptide ◽

Bacterial Resistance ◽

Cell Types ◽

Polymyxin B ◽

Sensor Kinase ◽

Multiple Cell ◽

Ob Fold ◽

Antimicrobial Peptide Resistance ◽

Multicellular Organisms

ABSTRACT Antimicrobial peptides (AMPs) kill or prevent the growth of microbes. AMPs are made by virtually all single and multicellular organisms and are encountered by bacteria in diverse environments, including within a host. Bacteria use sensor-kinase systems to respond to AMPs or damage caused by AMPs. Salmonella enterica deploys at least three different sensor-kinase systems to modify gene expression in the presence of AMPs: PhoP-PhoQ, PmrA-PmrB, and RcsB-RcsC-RcsD. The ydeI gene is regulated by the RcsB-RcsC-RcsD pathway and encodes a 14-kDa predicted oligosaccharide/oligonucleotide binding-fold (OB-fold) protein important for polymyxin B resistance in broth and also for virulence in mice. We report here that ydeI is additionally regulated by the PhoP-PhoQ and PmrA-PmrB sensor-kinase systems, which confer resistance to cationic AMPs by modifying lipopolysaccharide (LPS). ydeI, however, is not important for known LPS modifications. Two independent biochemical methods found that YdeI copurifies with OmpD/NmpC, a member of the trimeric β-barrel outer membrane general porin family. Genetic analysis indicates that ompD contributes to polymyxin B resistance, and both ydeI and ompD are important for resistance to cathelicidin antimicrobial peptide, a mouse AMP produced by multiple cell types and expressed in the gut. YdeI localizes to the periplasm, where it could interact with OmpD. A second predicted periplasmic OB-fold protein, YgiW, and OmpF, another general porin, also contribute to polymyxin B resistance. Collectively, the data suggest that periplasmic OB-fold proteins can interact with porins to increase bacterial resistance to AMPs.

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