Outer Membrane Protease Research Articles

Probing marine macroalgal phlorotannins as an antibacterial candidate against Salmonella typhi: Molecular docking and dynamics simulation approach

The increasing prevalence of drug-resistant bacterial strains, including multidrug-resistant Salmonella typhi, has raised significant concerns about the effectiveness of traditional antimicrobial treatments. To address this issue, the study employed computational techniques to evaluate the potential of natural phlorotannins derived from marine algae as alternative antibacterial agents against S. typhi. A total of 104 phlorotannins were retrieved from PubChem and subjected to molecular docking with three specific target proteins of S. typhi. Among the compounds tested, Compound-10 a derivative of dieckol exhibited the highest affinity for the Omptin family outer membrane protease [pgtE] protein and for the cell invasion protein sipB protein. The docking results revealed strong interactions between the phlorotannins and the target proteins, indicating their potential as antimicrobial agents. Additionally, pharmacokinetic studies using the QikProp module demonstrated that the top-ranked compounds showed favourable drug-like properties with good druggable efficiency, moderate gut-blood barrier transport, and high human oral absorption. Most compounds passed the rule of three and five drug-likeness criteria, indicating their potential as drug candidates. Furthermore, MM-GBSA analysis provided relative binding affinity estimations, ranking the compounds based on their calculated binding energies. These results align reasonably well with experimental binding affinities, reinforcing the potential of the identified compounds as potent binders to their target proteins. This study highlights the promising antibacterial potential of marine phlorotannins against S. typhi. Further in vitro studies are warranted to validate these compounds' efficacy and anti-microbial activity, ultimately paving the way for developing new therapeutic strategies to combat drug-resistant Salmonella infections.

Phase-Separating Peptides Recruiting Aggregation-Induced Emission Fluorogen for Rapid E. coli Detection.

Rationally designed biomolecular condensates have found applications primarily as drug-delivery systems, thanks to their ability to self-assemble under physico-chemical triggers (such as temperature, pH, or ionic strength) and to concomitantly trap client molecules with exceptionally high efficiency (>99%). However, their potential in (bio)sensing applications remains unexplored. Here, we describe a simple and rapid assay to detect E. coli by combining phase-separating peptide condensates containing a protease recognition site, within which an aggregation-induced emission (AIE)-fluorogen is recruited. The recruited AIE-fluorogen's fluorescence is easily detected with the naked eye when the samples are viewed under UV-A light. In the presence of E. coli, the bacteria's outer membrane protease (OmpT) cleaves the phase-separating peptides at the encoded protease recognition site, resulting in two shorter peptide fragments incapable of liquid-liquid phase separation. As a result, no condensates are formed and the fluorogen remains non-fluorescent. The assay feasibility was first tested with recombinant OmpT reconstituted in detergent micelles and subsequently confirmed with E. coli K-12. In its current format, the assay can detect E. coli K-12 (108 CFU) within 2 h in spiked water samples and 1-10 CFU/mL with the addition of a 6-7 h pre-culture step. In comparison, most commercially available E. coli detection kits can take anywhere from 8 to 24 h to report their results. Optimizing the peptides for OmpT's catalytic activity can significantly improve the detection limit and assay time. Besides detecting E. coli, the assay can be adapted to detect other Gram-negative bacteria as well as proteases having diagnostic relevance.

Salmonella Typhimurium PgtE is an essential arsenal to defend against the host resident antimicrobial peptides

Salmonella enterica serovar Typhimurium is a common cause of gastroenteritis in humans and occasionally causes systemic infection. Salmonella's ability to survive and replicate within macrophages is an important characteristic during systemic infection. The outer membrane protease PgtE of S. enterica is a member of the Omptin family of outer membrane aspartate proteases which has well-characterized proteolytic activities in-vitro against a wide range of physiologically relevant substrates. However, no study has been done so far that draws a direct correlation between these in-vitro observations and the biology of the pathogen in-vivo. The main goals of this study were to characterize the pathogenesis-associated functions of pgtE and study its role in the intracellular survival and in-vivo virulence of Salmonella Typhimurium. Our study elucidated a possible role of Salmonella Typhimurium pgtE in combating host antimicrobial peptide- bactericidal/ permeability increasing protein (BPI) to survive in human macrophages. The pgtE-deficient strain of Salmonella showed attenuated proliferation and enhanced colocalization with BPI in U937 and Thp1 cells. In the presence of polymixin B, the attenuated in-vitro survival of STM ΔpgtE suggested a role of PgtE against the antimicrobial peptides. In addition, our study revealed that compared to the wild type Salmonella, the pgtE mutant is replication-deficient in C57BL/6 mice. Further, we showed that PgtE interacts directly with several antimicrobial peptides (AMPs) in the host gut. This gives the pathogen a survival advantage and helps to mount a successful infection in the host

Rapid Detection of Escherichia coli: Optimized Peptide-Polythiophene Interactions Help Reduce Assay Time and Improve Naked-Eye Detection.

Recent improvements in methods for rapid detection of microbial contamination in food and water samples have aided in the development of on-site and point-of-care testing. Early detection, made possible via on-site testing, can help limit the spread of food and waterborne illnesses. Recently, we reported a novel fluorescence-based Omptin-Polythiophene assay (the assay) to detect Escherichia coli in contaminated water samples. The assay targets OmpT─an E. coli outer membrane protease─and exploits the protease's ability to cleave at dibasic sites within a peptide. By combining a peptide substrate optimized for OmpT with a conjugated polythiophene reporter molecule whose optical properties vary upon interaction with the intact or cleaved peptide, we demonstrated the detection of 1-10 CFU/mL and 105 CFU/mL E. coli in 5.5 and 1 h, respectively. In comparison, most microbial detection methods that rely on culturing and plating techniques take anywhere between 8 and 24 h to report their results. Herein we report significant improvements in the assay which include reducing the assay time from an already short 1 h to a mere 10 min for detecting E. coli in highly contaminated samples and augmenting the assay with colorimetric sensing capability for naked eye discernment under normal visible light or under UV-A light. These improvements were made possible by characterizing the optical changes resulting from the interaction of the peptide with five carboxylate-functionalized polythiophene variants carrying different 3- side chain carboxylic acids and by identifying preferential peptide substrates via the screening of ten peptide sequence variants for OmpT activity.

Amphiphilic Membrane Environments Regulate Enzymatic Behaviors of Salmonella Outer Membrane Protease.

The role of an amphiphilic environment in the functional regulation of integral membrane proteins is well appreciated but how specific amphiphilic surrounding influences the conformational plasticity and function of a protein is less obvious. We focus on the Salmonella phosphoglycerate transport system (pgt)-encoded outer membrane protease E (PgtE), which plays an important role in tissue infiltration and survival of Salmonella enterica. Despite our understanding of its physiological functions, elucidation of its enzymatic behavior in response to the immediate amphiphilic surrounding is lacking. We monitor the proteolytic activity of PgtE reconstituted in Zwittergent 3-12 detergent micelles or a 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayer and examine factors that influence its activity. We find, to our surprise, that PgtE, which is thought to elicit a rapid response toward various substrates, showed hysteretic enzymatic behavior, characterized by a prominent lag phase prior to achieving the exponential steady state in its detergent-stabilized form as well as in the outer membrane embedded native state in live bacteria. The lag phase was abolished under three conditions: preformation of an inactive detergent-stabilized PgtE-substrate complex without lipopolysaccharide (LPS), LPS-bound detergent-stabilized PgtE that had reached steady state velocity, or PgtE reconstituted into a POPC bilayer environment. Interestingly, detergent- and bilayer-stabilized PgtE showed comparable steady-state activity. And strikingly, lipopolysaccharide (LPS) becomes nonessential for the activation of PgtE when the protein is reconstituted in the phospholipid bilayer, contrasting a long-standing notion that LPS is required for proteases belonging to the omptin family to be proteolytically active. These findings suggest intriguing biological nuances for the proteolytic function of PgtE that were not well appreciated previously and offer new perspectives that may generally be applicable for omptins.

Magneto-controlled potentiometric assay for E. coli based on cleavage of peptide by outer-membrane protease T

Rapid, sensitive and reliable Escherichia coli (E. coli) detection and identification are critically important to protect public health. Here, we describe a magneto-controlled potentiometric assay for specific detection of E. coli cells by making use of the E. coli outer-membrane protease T (OmpT). OmpT is an endopeptidase that specifically cleaves peptide at dibasic sites. A rationally designed peptide serving as both OmpT substrate and potentiometric signal reporter was immobilized on magnetic beads. The rapid accumulation and extraction of peptide-functionalized magnetic beads on a polymeric membrane doped with an ion exchanger can be achieved using a magnetic force. The magnetic-field-assisted extraction of the peptide into the polymeric membrane ion-sensitive sensor, as confirmed by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, can lead to a rapid, stable and reproducible potential change. OmpT is capable of cleaving the positively charged peptide on the magnetic beads, thus resulting in charge density change. The change in charge density and subsequently the potential change can be readily detected and used for quantification of E. coli at levels down to 5.0 × 103 CFU mL−1. This work provides a versatile, rapid and reliable potentiometric method for E. coli detection.

In Silico Structural and Functional Characterization of HtrA Proteins of Leptospira spp.: Possible Implications in Pathogenesis.

Leptospirosis is a zoonosis caused by the pathogenic bacteria of the genus Leptospira. The identification of conserved outer membrane proteins among pathogenic strains is a major research target in elucidating mechanisms of pathogenicity. Surface-exposed proteins are most probably the ones involved in the interaction of leptospires with the environment. Some spirochetes use outer membrane proteases as a way to penetrate host tissues. HtrA is a family of proteins found in various cell types, from prokaryotes to primates. They are a set of proteases usually composed of a serine protease and PDZ domains, and they are generally transported to the periplasm. Here, we identified four genes—annotated as HtrA, LIC11111, LIC20143, LIC20144 and LIC11037—and another one annotated as a serine protease, LIC11112. It is believed that the last forms a functional heterodimer with LIC11111, since they are organized in one operon. Our analyses showed that these proteins are highly conserved among pathogenic strains. LIC11112, LIC20143, and LIC11037 have the serine protease domain with the conserved catalytic triad His-Asp-Ser. This is the first bioinformatics analysis of HtrA proteins from Leptospira that suggests their proteolytic activity potential. Experimental studies are warranted to elucidate this possibility.

Yersinia pestis Plasminogen Activator.

The Gram-negative bacterium Yersinia pestis causes plague, a fatal flea-borne anthropozoonosis, which can progress to aerosol-transmitted pneumonia. Y. pestis overcomes the innate immunity of its host thanks to many pathogenicity factors, including plasminogen activator, Pla. This factor is a broad-spectrum outer membrane protease also acting as adhesin and invasin. Y. pestis uses Pla adhesion and proteolytic capacity to manipulate the fibrinolytic cascade and immune system to produce bacteremia necessary for pathogen transmission via fleabite or aerosols. Because of microevolution, Y. pestis invasiveness has increased significantly after a single amino-acid substitution (I259T) in Pla of one of the oldest Y. pestis phylogenetic groups. This mutation caused a better ability to activate plasminogen. In paradox with its fibrinolytic activity, Pla cleaves and inactivates the tissue factor pathway inhibitor (TFPI), a key inhibitor of the coagulation cascade. This function in the plague remains enigmatic. Pla (or pla) had been used as a specific marker of Y. pestis, but its solitary detection is no longer valid as this gene is present in other species of Enterobacteriaceae. Though recovering hosts generate anti-Pla antibodies, Pla is not a good subunit vaccine. However, its deletion increases the safety of attenuated Y. pestis strains, providing a means to generate a safe live plague vaccine.

Redefining enteroaggregative Escherichia coli (EAEC): Genomic characterization of epidemiological EAEC strains.

Although enteroaggregative E. coli (EAEC) has been implicated as a common cause of diarrhea in multiple settings, neither its essential genomic nature nor its role as an enteric pathogen are fully understood. The current definition of this pathotype requires demonstration of cellular adherence; a working molecular definition encompasses E. coli which do not harbor the heat-stable or heat-labile toxins of enterotoxigenic E. coli (ETEC) and harbor the genes aaiC, aggR, and/or aatA. In an effort to improve the definition of this pathotype, we report the most definitive characterization of the pan-genome of EAEC to date, applying comparative genomics and functional characterization on a collection of 97 EAEC strains isolated in the course of a multicenter case-control diarrhea study (Global Enteric Multi-Center Study, GEMS). Genomic analysis revealed that the EAEC strains mapped to all phylogenomic groups of E. coli. Circa 70% of strains harbored one of the five described AAF variants; there were no additional AAF variants identified, and strains that lacked an identifiable AAF generally did not have an otherwise complete AggR regulon. An exception was strains that harbored an ETEC colonization factor (CF) CS22, like AAF a member of the chaperone-usher family of adhesins, but not phylogenetically related to the AAF family. Of all genes scored, sepA yielded the strongest association with diarrhea (P = 0.002) followed by the increased serum survival gene, iss (p = 0.026), and the outer membrane protease gene ompT (p = 0.046). Notably, the EAEC genomes harbored several genes characteristically associated with other E. coli pathotypes. Our data suggest that a molecular definition of EAEC could comprise E. coli strains harboring AggR and a complete AAF(I-V) or CS22 gene cluster. Further, it is possible that strains meeting this definition could be both enteric bacteria and urinary/systemic pathogens.

Outer-Membrane Protease (OmpT) Based E. coli Sensing with Anionic Polythiophene and Unlabeled Peptide Substrate.

E. coli and Salmonella are two of the most common bacterial pathogens involved in foodborne and waterborne related deaths. Hence, it is critical to develop rapid and sensitive detection strategies for near-outbreak applications. Reported is a simple and specific assay to detect as low as 1 CFU mL-1 of E. coli in water within 6 hours by targeting the bacteria's surface protease activity. The assay relies on polythiophene acetic acid (PTAA) as an optical reporter and a short unlabeled peptide (LL37FRRV ) previously optimized as a substrate for OmpT, an outer-membrane protease on E. coli. LL37FRRV interacts with PTAA to enhance its fluorescence while also inducing the formation of a helical PTAA-LL37FRRV construct, as confirmed by circular dichroism. However, in the presence of E. coli LL37FRRV is cleaved and can no longer affect the conformations and optical properties of PTAA. This ability to distinguish between an intact and cleaved peptide was investigated in detail using LL37FRRV sequence variants.

Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding.

Outer membrane protease (OmpT) is a 33.5 kDa aspartyl protease that cleaves at dibasic sites and is thought to function as a defense mechanism for E. coli against cationic antimicrobial peptides secreted by the host immune system. Despite carrying three dibasic sites in its own sequence, there is no report of OmpT autoproteolysis in vivo. However, recombinant OmpT expressed in vitro as inclusion bodies has been reported to undergo autoproteolysis during the refolding step, thus resulting in an inactive protease. In this study, we monitor and compare levels of in vitro autoproteolysis of folded and unfolded OmpT and examine the role of lipopolysaccharide (LPS) in autoproteolysis. SDS-PAGE data indicate that it is only the unfolded OmpT that undergoes autoproteolysis while the folded OmpT remains protected and resistant to autoproteolysis. This selective susceptibility to autoproteolysis is intriguing. Previous studies suggest that LPS, a co-factor necessary for OmpT activity, may play a protective role in preventing autoproteolysis. However, data presented here confirm that LPS plays no such protective role in the case of unfolded OmpT. Furthermore, OmpT mutants designed to prevent LPS from binding to its putative LPS-binding motif still exhibited excellent protease activity, suggesting that the putative LPS-binding motif is of less importance for OmpT’s activity than previously proposed.

Structural investigation of APRs to improve the solubility of outer membrane protease (PgtE) from Salmonella enterica serotype typhi- A multi-constraint approach

Outer membrane proteins were playing a crucial role on the several functions controlled by cell membranes even though they are not naturally expressed at higher levels. In order to obtain biologically active protein, the denaturation of these inclusion bodies must be optimized using chaotropic agents. Hence, this study focuses on improving the yield of Outer Membrane Protease (PgtE) from Salmonella enterica serotype Typhi (S. Typhi) using chaotropes and additives. Denaturation methods were tried with various pH, detergents, and reducing agents were used to optimize the solubility of PgtE with biologically active form. Due to the aggregation, we failed to achieve the maximum yield of PgtE. Consequently, we predicted 9 Aggregation Prone Regions (APRs) in PgtE, which are mutated by known structural Gatekeepers. We calculated the Aggregation Index (AI) of PgtE with 10 mM of aspartic acid as an additive in optimized buffer. In addition, the mutations at specific positions within the protein structure can act as APRs suppressors without affecting protein stability with CABS flex dynamics. The multiple sequence analysis demonstrate that aspartic acid is appropriate denaturing additive for other Gram-negative pathogens of Omptin family.

Outer Membrane Protease OmpT-Based Strategy for Simplified Analysis of Histone Post-Translational Modifications by Mass Spectrometry.

Histone modifications play an important role in regulating transcriptional gene expression and chromatin processes in eukaryotes. Increasing researches proved that aberrant post-translational modifications (PTMs) of histones is associated with many diseases. However, MS-based identification and quantification of histone PTMs are still challenging. Although classic chemical derivatization in conjunction with trypsin digestion is widely used for histone PTMs analysis in a bottom-up strategy, several side reactions have been observed in practice. In this work, outer membrane protease T (OmpT) was utilized as a protease for direct histone proteolysis and generated appropriate lengths of histone peptides for retention on reversed-phase chromatography. The powerful and unique tolerance of OmpT for modified lysines and arginines was demonstrated and can be quantitatively described for the first time, making it useful for detecting natural modifications. Using the optimized digestion conditions, we succeeded in identifying 121 histone marks from HEK293T cells, 42 of which were previously unreported. Additionally, histone H3 PTMs were quantitatively profiled in the KMS11 multiple myeloma cells and NSD2 selective knockout KMS11cells, revealing that NSD2 was of high specificity on H3K36 dimethylation. Histone chemical derivatizations are not required in our strategy, showing a remarkable strength over the conventional trypsin-based workflow.

Bacterial predator-prey coevolution accelerates genome evolution and selects on virulence-associated prey defences

Generalist bacterial predators are likely to strongly shape many important ecological and evolutionary features of microbial communities, for example by altering the character and pace of molecular evolution, but investigations of such effects are scarce. Here we report how predator-prey interactions alter the evolution of fitness, genomes and phenotypic diversity in coevolving bacterial communities composed of Myxococcus xanthus as predator and Escherichia coli as prey, relative to single-species controls. We show evidence of reciprocal adaptation and demonstrate accelerated genomic evolution specific to coevolving communities, including the rapid appearance of mutator genotypes. Strong parallel evolution unique to the predator-prey communities occurs in both parties, with predators driving adaptation at two prey traits associated with virulence in bacterial pathogens—mucoidy and the outer-membrane protease OmpT. Our results suggest that generalist predatory bacteria are important determinants of how complex microbial communities and their interaction networks evolve in natural habitats.

Development of a novel bacterial surface display system using truncated OmpT as an anchoring motif.

An efficient bacterial surface display system based on the anchoring motif derived from Escherichia coli (E. coli) outer membrane protease OmpT was developed in this study. Referring to the classical Lpp-OmpA (LOA) display system, the signal peptide and nine amino acids of mature Lpp were fused to the transmembrane domain comprising five β-strands of truncated OmpT to generate a novel Lpp-OmpT (LOT) display system. The C-terminal fusion strategy was used to fuse a small peptide (His tag) and red fluorescent protein (mCherry) to the C-terminus of LOT. Cell surface exposure of His tag and mCherry were compared between the LOA and LOT display systems. E. coli expressing LOT-His tag adsorbed more Cu2+ than E. coli expressing LOA-His tag. E. coli expressing both LOT-mCherry-His tag and LOA-mCherry-His tag adhered to Cu2+ chelating sepharose beads, and adhered cells could be dissociated from the beads after excess Cu2+ treatment. More importantly, compared with the LOA system, a higher amount of LOT-mCherry-His tag hybrid protein was demonstrated to be localized at the outer membrane by both fluorescence spectrophotometric determination of cell fractions and cell-surface immunofluorescence assay. These results suggest that genetically modified OmpT can be used as a potential anchoring motif to efficiently and stably display polypeptides and proteins, and that the LOT system could be used in a variety of biotechnological and industrial processes.

Substrate specificities in Salmonella typhi outer membrane protease (PgtE) from omptin family – An in silico proteomic approach

Salmonella typhi is a Gram-negative pathogen that causes severe systemic infections like typhoid fever and gastrointestinal disease in humans. PgtE belongs to the Omptin family, and plays a central role at the host-pathogen interface. In the present study, the S. typhi PgtE sequence was retrieved and the cleavage sites were predicted. Owing to the non-existence of a crystalline structure of S. typhi PgtE, theoretical 3D modeled structures were predicted using the I-TASSER server, which helps to understand the protease inhibitory mechanism. Molecular interaction studies with known protease inhibitors revealed that aspartic protease inhibitor Indinavir has the best interaction with S. typhi PgtE. From the metal ion docking studies, Mg2+ has better interaction compared to the Zn2+ and Cu2+ ions. The multiple pathogen sequence alignment of the Omptin proteases family shows that interacting residues were conserved among the Omptins. These results will provide new knowledge for the development of novel therapeutic strategies against S. typhi PgtE and Omptin family proteases.

In Silico Characterization of B Cell and T Cell Epitopes for Subunit Vaccine Design of Salmonella typhi PgtE: A Molecular Dynamics Simulation Approach.

Typhoid fever is an acute illness in humans, caused by Salmonella typhi, a gram-negative bacterium. Outer membrane proteins of S. typhi have strong potential for its use in the development of subunit vaccine against typhoid. In the current study, peptide-based subunit vaccine was constructed from outer membrane protease E (PgtE) against S. typhi. B cell and T cell epitopes were identified at fold level with a validated three-dimensional modeled structure. T cell epitopes from PgtE (IHPDTSANY) have 99.5% binding to a maximum number of major histocompatibility complex class I and class II alleles. They also bind to the typhoid-resistant human leukocyte antigen (HLA) alleles DRB1*0401. PgtE epitopes were docked with HLA-DR4 (PDB ID: 1D5M) and a contact map was constructed. A simulation search for the binding site for full flexibility of the peptide from CABS- (Cα, Cβ, side-chain)-dock shows stable interactions. Molecular dynamics simulation studies revealed that the PgtE-epitope complex structure was more stable throughout the simulation (20 ns) and interaction did not change the radius of gyration. In conclusion, computational analysis, molecular docking, and molecular dynamics (MD) simulation of PgtE-epitope complex were used to elucidate the binding mode, and the dynamical changes of epitopes were more suitable for vaccine development against typhoid.

Unexpected transcriptome pompTʹ contributes to the increased pathogenicity of a pompT mutant of avian pathogenic Escherichia coli

The lambda red recombination system makes it suitable for screening virulence gene utility in avian pathogenic Escherichia coli (APEC) on account of its wide applicability, simplicity and high efficiency. In APEC E058 (O2 serogroup), there are two copies of the outer membrane protease (ompT) gene, compT encoding cOmpT that is located on the chromosome and pompT encoding pOmpT that is located on a ColV plasmid. However, the relationship between pathogenesis and pompT expression in APEC E058 has yet to be elucidated. Here, we successfully constructed two pompT gene mutants: E058ΔpompT containing a chloramphenicol (cat) resistance gene and E058ΔpompTʹ without the cat gene. By RT-PCR and sequencing analysis, an unexpected transcriptome pompTʹ was detected in mutant strain E058ΔpompTʹ after deletion of the cat gene induced by the lambda red recombination system. Surprisingly, the pathogenicity of mutant E058ΔpompT was significantly attenuated compared to its parental strain in the chicken infection model and HD11 cell model then the pompT gene was knocked out, while the pathogenicity of the other mutant strain E058ΔpompTʹ had no difference. Furthermore, the presence of unexpected transcriptome pompTʹ influenced the bactericidal activity of SPF chicken serum and decreased the transcription level of TLR2 in the heart tissue of chickens. Our study identifies the pompT gene plays an important role in the virulence of APEC E058, and the unexpected transcriptome pompTʹ contributes to the increased pathogenicity of APEC E058 mutants following deletion of the cat gene induced by the lambda red recombination system, which suggests that this system still has some limitations for construction of mutant strains particularly where these are used in development of live vaccine.

The Role of Outer Membrane Proteins and Lipopolysaccharides for the Sensitivity of Escherichia coli to Antimicrobial Peptides

Bacterial resistance to classical antibiotics is emerging worldwide. The number of infections caused by multidrug resistant bacteria is increasing and becoming a serious threat for human health globally. In particular, Gram-negative pathogens including multidrug resistant Escherichia coli are of serious concern being resistant to the currently available antibiotics. All Gram-negative bacteria are enclosed by an outer membrane which acts as an additional protection barrier preventing the entry of toxic compounds including antibiotics and antimicrobial peptides (AMPs). In this study we report that the outer membrane component lipopolysaccharide (LPS) plays a crucial role for the antimicrobial susceptibility of E. coli BW25113 against the cationic AMPs Cap18, Cap11, Cap11-1-18m2, melittin, indolicidin, cecropin P1, cecropin B, and the polypeptide antibiotic colistin, whereas the outer membrane protease OmpT and the lipoprotein Lpp only play a minor role for the susceptibility against cationic AMPs. Increased susceptibility toward cationic AMPs was found for LPS deficient mutants of E. coli BW25113 harboring deletions in any of the genes required for the inner part of core-oligosaccharide of the LPS, waaC, waaE, waaF, waaG, and gmhA. In addition, our study demonstrates that the antimicrobial activity of Cap18, Cap11, Cap11-1-18m2, cecropin B, and cecropin P1 is not only dependent on the inner part of the core oligosaccharide, but also on the outer part and its sugar composition. Finally, we demonstrated that the antimicrobial activity of selected Cap18 derivatives harboring amino acid substitutions in the hydrophobic interface, are non-active against wild-type E. coli ATCC29522. By deleting waaC, waaE, waaF, or waaG the antimicrobial activity of the non-active derivatives can be partially or fully restored, suggesting a very close interplay between the LPS core oligosaccharide and the specific Cap18 derivative. Summarizing, this study implicates that the nature of the outer membrane component LPS has a big impact on the antimicrobial activity of cationic AMPs against E. coli. In particular, the inner as well as the outer part of the core oligosaccharide are important elements determining the antimicrobial susceptibility of E. coli against cationic AMPs.

Humoral and cellular immune responses to Yersinia pestis Pla antigen in humans immunized with live plague vaccine.

BackgroundTo establish correlates of human immunity to the live plague vaccine (LPV), we analyzed parameters of cellular and antibody response to the plasminogen activator Pla of Y. pestis. This outer membrane protease is an essential virulence factor that is steadily expressed by Y. pestis.Methodology/Principal findingsPBMCs and sera were obtained from a cohort of naïve (n = 17) and LPV-vaccinated (n = 34) donors. Anti-Pla antibodies of different classes and IgG subclasses were determined by ELISA and immunoblotting. The analysis of antibody response was complicated with a strong reactivity of Pla with normal human sera. The linear Pla B-cell epitopes were mapped using a library of 15-mer overlapping peptides. Twelve peptides that reacted specifically with sera of vaccinated donors were found together with a major cross-reacting peptide IPNISPDSFTVAAST located at the N-terminus. PBMCs were stimulated with recombinant Pla followed by proliferative analysis and cytokine profiling. The T-cell recall response was pronounced in vaccinees less than a year post-immunization, and became Th17-polarized over time after many rounds of vaccination.Conclusions/SignificanceThe Pla protein can serve as a biomarker of successful vaccination with LPV. The diagnostic use of Pla will require elimination of cross-reactive parts of the antigen.