Omptin Proteases of Enterobacterales Show Conserved Regulation by the PhoPQ Two-Component System but Exhibit Divergent Protection from Antimicrobial Host Peptides and Complement.

Bacteria that colonize eukaryotic surfaces interact with numerous antimicrobial host-produced molecules, including host defense peptides, complement, and antibodies. Bacteria have evolved numerous strategies to both detect and resist these molecules, and in the Enterobacterales order of bacteria these include alterations of the cell surface lipopolysaccharide structure and/or charge and the production of proteases that can degrade these antimicrobial molecules. Here, we show that omptin family proteases from Escherichia coli and Citrobacter rodentium are regulated by the PhoPQ system. Omptin protease activity is induced by growth in low Mg2+, and deletion of PhoP dramatically reduces omptin protease activity, transcriptional regulation, and protein levels. We identify conserved PhoP-binding sites in the promoters of the E. coli omptin genes ompT, ompP, and arlC as well as in croP of Citrobacter rodentium and show that mutation of the putative PhoP-binding site in the ompT promoter abrogates PhoP-dependent expression. Finally, we show that although regulation by PhoPQ is conserved, each of the omptin proteins has differential activity toward host defense peptides, complement components, and resistance to human serum, suggesting that each omptin confers unique survival advantages against specific host antimicrobial factors.

E. coli outer membrane protein T (OmpT) nanopore for peptide sensing.

Peptide detection methods with facility and high sensitivity are essential for diagnosing disease associated with peptide biomarkers. Nanopore sensing technology had emerged as a low cost, high-throughput, and scalable tool for peptide detection. The omptins family proteins which can form ß-barrel pores have great potentials to be developed as nanopore biosensor. However, there are no study about the channel properties of E. coli OmpT and the development of OmpT as a nanopore biosensor. In this study, the OmpT biological nanopore channel was constructed with a conductance of 1.49 nS in 500 mM NaCl buffer and a three-step gating phenomenon under negative voltage higher than 100 mV and then was developed as a peptide biosensor which can detect peptide without the interfere of ssDNA and dNTPs. The OmpT constructed in this study has potential application in peptide detection, and also provides a new idea for the detection of peptides using the specific binding ability of protease.

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

OBJECTIVES: 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. RESULTS: 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 ß-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. CONCLUSIONS: 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.

Enterohaemorrhagic Escherichia coli produces outer membrane vesicles as an active defence system against antimicrobial peptide LL-37.

Antimicrobial peptides (AMPs) are important components of the innate immune system. Enterohaemorrhagic Escherichia coli (EHEC), a food-borne pathogen causing serious diarrheal diseases, must overcome attack by AMPs. Here, we show that resistance of EHEC against human cathelicidin LL-37, a primary AMP, was enhanced by butyrate, which has been shown to act as a stimulant for the expression of virulence genes. The increase of resistance depended on the activation of the ompT gene, which encodes the outer membrane protease OmpT for LL-37. The expression of the ompT gene was enhanced through the activation system for virulence genes. The increase in ompT expression did not result in an increase in OmpT protease in bacteria but in enhancement of the production of OmpT-loaded outer membrane vesicles (OMVs), which primarily contributed to the increase in LL-37-resistance. Furthermore, a sublethal dosage of LL-37 stimulated the production of OMVs. Finally, we showed that OMVs produced by OmpT-positive strains protect the OmpT-negative strain, which is susceptible to LL-37 by itself more efficiently than OMVs from the ompT mutant. These results indicate that EHEC enhances the secretion of OmpT-loaded OMVs in coordination with the activation of virulence genes during infection and blocks bacterial cell attack by LL-37.

Distribution of virulence genes and multiple drug-resistant patterns amongst different phylogenetic groups of uropathogenic Escherichia coli isolated from patients with urinary tract infection.

A total of 85 Uropathogenic Escherichia coli (UPEC) isolates were screened against ceftiofur, oxacillin, nitrofurantoin and lincospectin using Kirby-Bauer disc diffusion method, following CLSI guidelines. Prevalence of virulent factor genes amongst the isolates was determined by PCR, using gene-specific primers against the different virulent factors. Statistical analysis of the data was performed using SPSS software. The prevalence of traT, ompT, Iss, malX and ibeA genes was 47.1%, 38.8%, 20%, 16.5% and 9.4%, respectively. The most prevalent gene in group A and D was traT, whilst in group B2 was Iss. The highest resistance has been shown against oxacillin (98.8%), followed by ceftiofur (77.6%), whilst resistance to lincospectin (2.4%) and nitrofurantoin (12.9%) had the lowest frequencies. Multidrug resistance was shown in 82.35% of the isolates, whilst this study recommend lincospectin and nitrofurantoin as choice drugs for treatment, but more investigation of the bacterial pathogenicity associated with urinary tract infection (UTI) may contribute to a better medical intervention.

Maximized Autotransporter-Mediated Expression (MATE) for Surface Display and Secretion of Recombinant Proteins in Escherichia coli.

A new optimized system for the surface display and secretion of recombinant proteins is described, termed MATE (maximized autotransporter-mediated expression). It is based on an artificial gene consisting of the coding region for the signal peptide of CtxB, a multiple cloning site for passenger gene insertion, flanked by coding sequences for linear epitopes for monoclonal antibodies and OmpT, and factor Xa protease cleavage sites followed by a codon-optimized DNA sequence of the linker and the ß-barrel of the type V autotransporter EhaA from Escherichia coli under control of an IPTG-inducible T5 promoter. The MATE system enabled the continuous secretion of recombinant passenger mCherry via OmpT-mediated cleavage, using native OmpT protease activity in E. coli when grown at 37 °C. It is the first example to show that native OmpT activity is sufficient to facilitate the secretion of a correctly folded target protein in preparative amounts obtaining 240 µg of purified mCherry from 800 mL of crude culture supernatant. Because the release of mCherry was achieved by a simple transfer of the encoding plasmid from an OmpT-negative to an OmpT-positive strain, it bears the option to use surface display for screening purposes and secretion for production of the selected variant. A single plasmid could therefore be used for continuous secretion in OmpT-positive strains or surface display in OmpT-negative strains. In conclusion, the MATE system appears to be a versatile tool for the surface display and for the secretion of target proteins in E. coli.

Orthopaedic Manual Physical Therapy: A Modern Definition and Description.

Currently, orthopaedic manual physical therapy (OMPT) lacks a description of practice that reflects contemporary thinking and embraces advances across the scientific, clinical, and educational arms of the profession. The absence of a clear definition of OMPT reduces understanding of the approach across health care professions and potentially limits OMPT from inclusion in scientific reviews and clinical practice guidelines. For example, it is often incorrectly classified as passive care or incorrectly contrasted with exercise-therapy approaches. This perspective aims to provide clinicians, researchers, and stakeholders a modern definition of OMPT that improves the understanding of this approach both inside and outside the physical therapist profession. The authors also aim to outline the unique and essential aspects of advanced OMPT training with the corresponding examination and treatment competencies. This definition of practice and illustration of its defining characteristics is necessary to improve the understanding of this approach and to help classify it correctly for study in the scientific literature. This perspective provides a current definition and conceptual model of OMPT, defining the distinguishing characteristics and key elements of this systematic and active patient-centered approach to improve understanding and help classify it correctly for study in the scientific literature.

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.

Outer Membrane Vesicles of Vibrio cholerae Protect and Deliver Active Cholera Toxin to Host Cells via Porin-Dependent Uptake.

Outer membrane vesicles (OMVs) are an emerging research field due to their multifactorial composition and involvement in interspecies and intraspecies communication. Recent studies indicate that vesicle release by Gram-negative bacterial pathogens is increased during in vivo colonization, as exemplified by the facultative human pathogen Vibrio cholerae upon oral ingestion by the host. In this study, we investigate the fate of OMVs produced by the Gram-negative facultative pathogen V. cholerae. We show that vesicles produced by the clinically relevant El Tor biotype are readily taken up by human intestinal cell lines. We identify outer membrane porins of V. cholerae, i.e., OmpU and OmpT, as the required surface effectors on OMVs for cellular uptake, and we pinpoint the uptake mechanism as caveolin-mediated endocytosis. Furthermore, we show that OMVs derived from V. cholerae grown under virulence-inducing conditions act as potent vehicles for delivery of bioactive cholera toxin to intestinal epithelial cells. In contrast to free cholera toxin secreted via the type II secretion system, OMV-associated cholera toxin is protected from degradation by intestinal proteases. Taken together, these data show that OMV-associated cholera toxin can sustain longer periods in the intestinal tract and preserve toxin effects, as indicated by a prolonged increase of cAMP levels in the intestinal tissue. IMPORTANCE Cholera is still a massive global health burden because it causes large outbreaks with millions of infections and thousands of deaths every year. Several studies have contributed to the knowledge of this pathogen, although key parts are still missing. We aim to broaden our understanding of Vibrio cholerae infections, virulence, and toxicity by drawing attention to the involvement of OMVs in these core processes. Upon host entry, V. cholerae increases secretion of OMVs, which can carry the main virulence factor, cholera toxin, to distant host intestinal cells. We show that specific outer membrane porins on the vesicle surface mediate endocytosis of the vesicles into intestinal cells. With protection by the vesicles, cholera toxin activity endures even in the presence of intestinal proteases. It is tempting to hypothesize that the extended half-life of vesicle-associated cholera toxin allows it to target host cells distant from the primary colonization sites.

Deciphering Additional Roles for the EF-Tu, l-Asparaginase II and OmpT Proteins of Shiga Toxin-Producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) causes outbreaks and sporadic cases of gastroenteritis. STEC O157:H7 is the most clinically relevant serotype in the world. The major virulence determinants of STEC O157:H7 are the Shiga toxins and the locus of enterocyte effacement. However, several accessory virulence factors, mainly outer membrane proteins (OMPs) that interact with the host cells may contribute to the virulence of this pathogen. Previously, the elongation factor thermo unstable (EF-Tu), l-asparaginase II and OmpT proteins were identified as antigens in OMP extracts of STEC. The known subcellular location of EF-Tu and l-asparaginase II are the cytoplasm and periplasm, respectively. Therefore, we investigate whether these two proteins may localize on the surface of STEC and, if so, what roles they have at this site. On the other hand, the OmpT protein, a well characterized protease, has been described as participating in the adhesion of extraintestinal pathogenic E. coli strains. Thus, we investigate whether OmpT has this role in STEC. Our results show that the EF-Tu and l-asparaginase II are secreted by O157:H7 and may also localize on the surface of this bacterium. EF-Tu was identified in outer membrane vesicles (OMVs), suggesting it as a possible export mechanism for this protein. Notably, we found that l-asparaginase II secreted by O157:H7 inhibits T-lymphocyte proliferation, but the role of EF-Tu at the surface of this bacterium remains to be elucidated. In the case of OmpT, we show its participation in the adhesion of O157:H7 to human epithelial cells. Thus, this study extends the knowledge of the pathogenic mechanisms of STEC.

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.

Outer Membrane Vesiculation Facilitates Surface Exchange and In Vivo Adaptation of Vibrio cholerae.

Gram-negative bacteria release outer membrane vesicles into the external milieu to deliver effector molecules that alter the host and facilitate virulence. Vesicle formation is driven by phospholipid accumulation in the outer membrane and regulated by the phospholipid transporter VacJ/Yrb. We use the facultative human pathogen Vibrio cholerae to show that VacJ/Yrb is silenced early during mammalian infection, which stimulates vesiculation that expedites bacterial surface exchange and adaptation to the host environment. Hypervesiculating strains rapidly alter their bacterial membrane composition and exhibit enhanced intestinal colonization fitness. This adaptation is exemplified by faster accumulation of glycine-modified lipopolysaccharide (LPS) and depletion of outer membrane porin OmpT, which confers resistance to host-derived antimicrobial peptides and bile, respectively. The competitive advantage of hypervesiculation is lost upon pre-adaptation to bile and antimicrobial peptides, indicating the importance of these adaptive processes. Thus, bacteria use outer membrane vesiculation to exchange cell surface components, thereby increasing survival during mammalian infection.

Identification and characterization of OmpT-like proteases in uropathogenic Escherichia coli clinical isolates.

Bacterial colonization of the urogenital tract is limited by innate defenses, including the production of antimicrobial peptides (AMPs). Uropathogenic Escherichia coli (UPEC) resist AMP-killing to cause a range of urinary tract infections (UTIs) including asymptomatic bacteriuria, cystitis, pyelonephritis, and sepsis. UPEC strains have high genomic diversity and encode numerous virulence factors that differentiate them from non-UTI-causing strains, including ompT. As OmpT homologs cleave and inactivate AMPs, we hypothesized that UPEC strains from patients with symptomatic UTIs have high OmpT protease activity. Therefore, we measured OmpT activity in 58 clinical E. coli isolates. While heterogeneous OmpT activities were observed, OmpT activity was significantly greater in UPEC strains isolated from patients with symptomatic infections. Unexpectedly, UPEC strains exhibiting the greatest protease activities harbored an additional ompT-like gene called arlC (ompTp). The presence of two OmpT-like proteases in some UPEC isolates led us to compare the substrate specificities of OmpT-like proteases found in E. coli. While all three cleaved AMPs, cleavage efficiency varied on the basis of AMP size and secondary structure. Our findings suggest the presence of ArlC and OmpT in the same UPEC isolate may confer a fitness advantage by expanding the range of target substrates.

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.

Unusual Constriction Zones in the Major Porins OmpU and OmpT from Vibrio cholerae.

The outer membranes (OM) of many Gram-negative bacteria contain general porins, which form nonspecific, large-diameter channels for the diffusional uptake of small molecules required for cell growth and function. While the porins of Enterobacteriaceae (e.g., E. coli OmpF and OmpC) have been extensively characterized structurally and biochemically, much less is known about their counterparts in Vibrionaceae. Vibrio cholerae, the causative agent of cholera, has two major porins, OmpU and OmpT, for which no structural information is available despite their importance for the bacterium. Here we report high-resolution X-ray crystal structures of V. cholerae OmpU and OmpT complemented with molecular dynamics simulations. While similar overall to other general porins, the channels of OmpU and OmpT have unusual constrictions that create narrower barriers for small-molecule permeation and change the internal electric fields of the channels. Together with electrophysiological and in vitro transport data, our results illuminate small-molecule uptake within the Vibrionaceae.

Role of outer membrane protein T in pathogenicity of avian pathogenic Escherichia coli.

An outer membrane protein T (OmpT) could play a vital role in the pathogenesis of the neonatal meningitis Escherichia coli (NMEC) in human and animals. However, whether ompT plays a role in avian pathogenic E. coli (APEC) infection remains unclear. In this study we evaluated the potential of ompT in APEC pathogenesis. An ompT gene was deleted from APEC mutant strain (TW-XM) was constructed and characterized. The inactivation of ompT reduced significantly the adherence and invasion capabilities of APEC to mouse brain microvascular endothelial cell (BMEC) bEnd.3 cells at the rates of 43.8% and 28.8% respectively, compared with the wild strain TW-XM. Further studies showed that deletion of ompT gene reduced the bacterial virulence with 15.2-fold in ducklings and 9.7-fold in mouse models based on the measurement of the LD50. Furthermore, experimental infection of animals revealed that, loss of ompT showed reduced APEC colonization and invasion capacity in brains, lungs and blood by 2-fold, 1.96-fold, and 1.7-fold, respectively, compared with the wild-type strain TW-XM. These virulence-related phenotypes were partially recoverable by genetic complementation. The results of the quantitative real-time reverse transcription-PCR (qRT-PCR) indicated that the loss of ompT significantly decreased the expression levels of ompA, fimC and tsh in the mutant strain ΔOmpT, when compared with TW-XM (p<0.01). Collectively, our data showed that inactivation of ompT decreased adhesion, invasion, colonization, proliferation capacities, possibly by reduced expression levels of ompA, fimC and tsh, which may justify that, ompT is implicated in APEC pathogenicity.

Role of uropathogenic Escherichia coli outer membrane protein T in pathogenesis of urinary tract infection.

OmpT is one of the members of the outer membrane protein family that has been identified as a virulence factor in most of the uropathogenic Escherichia coli (UPEC). However, the exact role of OmpT in the urinary tract infections (UTIs) remains unclear. To determine the role of OmpT in the pathogenesis of UPEC, an isogenic deletion mutant of ompT (COTD) was constructed by the λ Red recombination. Human bladder epithelial cell line 5637(HBEC 5637) was used to evaluate the ability of bacterial adhesion/invasion. A murine model of UTI was established to study the formation of intracellular bacterial communities (IBCs) in the process of UTIs. The cytokines were also examined during the pathogenesis. The results showed that the COTD strain was deficient in bacterial adhesion and invasion as well as in IBC formation compare to the parent strain. ELISA quantification analysis of cytokines showed that the levels of TNF-α, IL-6 and IL-8 in the serum, bladder and kidney tissues of the mice infected with COTD were lower than that of the CFT073 group. In summary, these results suggest that OmpT plays a multifaceted role in pathogenesis of UTI, including increased bacterial adhesiveness/invasiveness, formation of IBCs and upregulated proinflammatory cytokines.

Study on the virulence of an ompT deletion mutant of Escherichia coli causing neonatal meningitis / 中华微生物学和免疫学杂志

Objective To investigate the virulence role of ompT of Escherichia coli in the patho-genesis of neonatal meningitis .Methods Adhesive abilities of the parent strain E 44 and the isogenic ompT-deletion mutant strain ( E44 ∶ΔompT) to human brain microvascular endothelial cells were evaluated in in vitro model.Low-copy-number plasmid pST containing ompT locus and point mutant plasmid pST 85 were transferred into E44 ∶ΔompT to construct the complemented mutant strain , and its adhesive ability was ana-lyzed.Influences of ompT deletion on E44 strain in its ability of bacterial intestinal colonization and ability of penetrating the blood-brain barrier were determined . Results In comparison with the parent strain , E44 ∶ΔompT strain showed significantly impaired adhesive ability to human brain microvascular endothelial cells, which could be partly restored by inserting the complementary plasmids of pST and pST 85.Deletion of the ompT did not affect Escherichia coli K1 in normal intestinal colonization in in vivo model.E44 ∶ΔompT strain could induce bacteremia , which was similar to that induced by the parent strain , but its ability of crossing the blood-brain barrier was significantly declined .Conclusion The study demonstrate that ompT plays an important role as the virulence element of Escherichia coli in binding to brain microvascular endothe-lial cells and penetrating the blood-brain barrier .Further study should be performed to investigate the influ-ences of OmpT proteinase on the virulence of Escherichia coil.