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Amino terminus mutant OmpA from an isolated antibiotic resistant Escherichia coli still possess resistance to environmental stresses  [PDF]
Zhiping Zhao, Xin Nie, Zaixin Li, Zhi Zhang, Jie Ding, Wanru Xie
Advances in Biological Chemistry (ABC) , 2013, DOI: 10.4236/abc.2013.31014
Abstract:

Antibiotic resistant Escherichia coli strains are becoming more common recently. OmpA is a very important antigen protein of E. coli, which consists of two separate domains, N-terminal and C-terminal domain. The N-terminal domain contains eight β- barrel regions that plays important roles in the multifaceted functions of OmpA. In the present study, we cloned a mutant OmpA gene from a multi-antibiotic resistant E. coli strain. Sequence analysis indicated that the N-terminal DNA sequence of the mutant OmpA shared 81.05% homology with the modeled OmpA from E. coli K12 and the N-terminal amino acid sequence of the mutant OmpA was 81.22% identical to that of the E. coli K12 OmpA. Moreover, several amino acids located in the β-barrel region were mutated. The mutant OmpA was expressed in BL21 suggested by SDS-PAGE. Resistance to environmental stress assay indicated that the N-terminus mutant OmpA still possessed excellent activities in pH, temperature and osmotic pressure resistance. Our pre- sent study may supply insights into better and deeper understand the relationships between OmpA N-terminal regions and its functions in environmental stress conditions and the mechanisms on antibiotic resistance of E. coli.

Block and Boost DNA Transfer: Opposite Roles of OmpA in Natural and Artificial Transformation of Escherichia coli  [PDF]
Dongchang Sun, Bing Wang, Lihong Zhu, Mengyao Chen, Linlin Zhan
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0059019
Abstract: Our previous work established that DNA is naturally transferable on agar plates through a new transformation system which is regulated by the stationary phase master regulator RpoS in Escherichia coli. In this transformation system, neither additional Ca2+ nor heat shock is required. Instead, transformation is stimulated by agar. The membrane protein OmpA, a gated pore permeable to ions and larger solutes, serves as a receptor for DNA transfer during bacteriophage infection and conjugation. However, it remains unknown how DNA transfers across membranes and whether OmpA is involved in transformation of E. coli. Here, we explored potential roles of OmpA in natural and chemical transformation of E. coli. We observed that ompA inactivation significantly improved natural transformation on agar plates, indicating that OmpA blocks DNA transfer. Transformation promotion by ompA inactivation also occurred on soft plates, indicating that OmpA blocks DNA transfer independent of agar. By contrast, compared with the wild-type strain, chemical transformation of the ompA mutant was lower, indicating that OmpA has a role in DNA transfer. Inactivation of ompA also reduced chemical transformation in solution containing less Ca2+ or with a shortened time for heat shock, suggesting that the promotion effect of OmpA on DNA transfer does not solely rely on Ca2+ or heat shock. We conclude that OmpA plays opposite roles in natural and chemical transformation: it blocks DNA uptake on agar plates but promotes DNA transfer in the liquid Ca2+ solution. Considering that no single factor was identified to reverse the function of OmpA, we propose that multiple factors may cooperate in the functional reversal of OmpA during natural and artificial transformation of E. coli. Finally, we observed that ompA transcription was not affected by the expression of RpoS, excluding the possibility that RpoS regulates DNA transfer by suppressing ompA transcription.
Outer Membrane Protein A (OmpA): A New Player in Shigella flexneri Protrusion Formation and Inter-Cellular Spreading  [PDF]
Cecilia Ambrosi, Monica Pompili, Daniela Scribano, Carlo Zagaglia, Sandro Ripa, Mauro Nicoletti
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0049625
Abstract: Outer membrane protein A (OmpA) is a multifaceted predominant outer membrane protein of Escherichia coli and other Enterobacteriaceae whose role in the pathogenesis of various bacterial infections has recently been recognized. Here, the role of OmpA on the virulence of Shigella flexneri has been investigated. An ompA mutant of wild-type S. flexneri 5a strain M90T was constructed (strain HND92) and it was shown to be severely impaired in cell-to-cell spreading since it failed to plaque on HeLa cell monolayers. The lack of OmpA significantly reduced the levels of IcsA while the levels of cell associated and released IcsP-cleaved 95 kDa amino-terminal portion of the mature protein were similar. Nevertheless, the ompA mutant displayed IcsA exposed across the entire bacterial surface. Surprisingly, the ompA mutant produced proper F-actin comet tails, indicating that the aberrant IcsA exposition at bacterial lateral surface did not affect proper activation of actin-nucleating proteins, suggesting that the absence of OmpA likely unmasks mature or cell associated IcsA at bacterial lateral surface. Moreover, the ompA mutant was able to invade and to multiply within HeLa cell monolayers, although internalized bacteria were found to be entrapped within the host cell cytoplasm. We found that the ompA mutant produced significantly less protrusions than the wild-type strain, indicating that this defect could be responsible of its inability to plaque. Although we could not definitely rule out that the ompA mutation might exert pleiotropic effects on other S. flexneri genes, complementation of the ompA mutation with a recombinant plasmid carrying the S. flexneri ompA gene clearly indicated that a functional OmpA protein is required and sufficient for proper IcsA exposition, plaque and protrusion formation. Moreover, an independent ompA mutant was generated. Since we found that both mutants displayed identical virulence profile, these results further supported the findings presented in this study.
Display of Multimeric Antimicrobial Peptides on the Escherichia coli Cell Surface and Its Application as Whole-Cell Antibiotics  [PDF]
Ju Ri Shin, Ki Jung Lim, Da Jung Kim, Ju Hyun Cho, Sun Chang Kim
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0058997
Abstract: Concerns over the increasing emergence of antibiotic-resistant pathogenic microorganisms due to the overuse of antibiotics and the lack of effective antibiotics for livestock have prompted efforts to develop alternatives to conventional antibiotics. Antimicrobial peptides (AMPs) with a broad-spectrum activity and rapid killing, along with little opportunity for the development of resistance, represent one of the promising novel alternatives. Their high production cost and cytotoxicity, however, limit the use of AMPs as effective antibiotic agents to livestock. To overcome these problems, we developed potent antimicrobial Escherichia coli displaying multimeric AMPs on the cell surface so that the AMP multimers can be converted into active AMP monomers by the pepsin in the stomach of livestock. Buf IIIb, a strong AMP without cytotoxicity, was expressed on the surface of E. coli as Lpp-OmpA-fused tandem multimers with a pepsin substrate residue, leucine, at the C-terminus of each monomer. The AMP multimers were successfully converted into active AMPs upon pepsin cleavage, and the liberated Buf IIIb-L monomers inhibited the growth of two major oral infectious pathogens of livestock, Salmonella enteritidis and Listeria monocytogenes. Live antimicrobial microorganisms developed in this study may represent the most effective means of providing potent AMPs to livestock, and have a great impact on controlling over pathogenic microorganisms in the livestock production.
Genes for the Major Structural Components of Thermotogales Species’ Togas Revealed by Proteomic and Evolutionary Analyses of OmpA and OmpB Homologs  [PDF]
Amanda K. Petrus, Kristen S. Swithers, Chaman Ranjit, Si Wu, Heather M. Brewer, J. Peter Gogarten, Ljiljana Pasa-Tolic, Kenneth M. Noll
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0040236
Abstract: The unifying structural characteristic of members of the bacterial order Thermotogales is their toga, an unusual cell envelope that includes a loose-fitting sheath around each cell. Only two toga-associated structural proteins have been purified and characterized in Thermotoga maritima: the anchor protein OmpA1 (or Ompα) and the porin OmpB (or Ompβ). The gene encoding OmpA1 (ompA1) was cloned and sequenced and later assigned to TM0477 in the genome sequence, but because no peptide sequence was available for OmpB, its gene (ompB) was not annotated. We identified six porin candidates in the genome sequence of T. maritima. Of these candidates, only one, encoded by TM0476, has all the characteristics reported for OmpB and characteristics expected of a porin including predominant β-sheet structure, a carboxy terminus porin anchoring motif, and a porin-specific amino acid composition. We highly enriched a toga fraction of cells for OmpB by sucrose gradient centrifugation and hydroxyapatite chromatography and analyzed it by LC/MS/MS. We found that the only porin candidate that it contained was the TM0476 product. This cell fraction also had β-sheet character as determined by circular dichroism, consistent with its enrichment for OmpB. We conclude that TM0476 encodes OmpB. A phylogenetic analysis of OmpB found orthologs encoded in syntenic locations in the genomes of all but two Thermotogales species. Those without orthologs have putative isofunctional genes in their place. Phylogenetic analyses of OmpA1 revealed that each species of the Thermotogales has one or two OmpA homologs. T. maritima has two OmpA homologs, encoded by ompA1 (TM0477) and ompA2 (TM1729), both of which were found in the toga protein-enriched cell extracts. These annotations of the genes encoding toga structural proteins will guide future examinations of the structure and function of this unusual lineage-defining cell sheath.
Differential Bacterial Surface Display of Peptides by the Transmembrane Domain of OmpA  [PDF]
Gertjan S. Verhoeven, Svetlana Alexeeva, Marileen Dogterom, Tanneke den Blaauwen
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0006739
Abstract: Peptide libraries or antigenic determinants can be displayed on the surface of bacteria through insertion in a suitable outer membrane scaffold protein. Here, we inserted the well-known antibody epitopes 3xFLAG and 2xmyc in exterior loops of the transmembrane (TM) domain of OmpA. Although these highly charged epitopes were successfully displayed on the cell surface, their levels were 10-fold reduced due to degradation. We verified that the degradation was not caused by the absence of the C-terminal domain of OmpA. In contrast, a peptide that was only moderately charged (SA-1) appeared to be stably incorporated in the outer membrane at normal protein levels. Together, these results suggest that the display efficiency is sensitive to the charge of the inserted epitopes. In addition, the high-level expression of OmpA variants with surface-displayed epitopes adversely affected growth in a strain dependent, transient manner. In a MC4100 derived strain growth was affected, whereas in MC1061 derived strains growth was unaffected. Finally, results obtained using a gel-shift assay to monitor β-barrel folding in vivo show that the insertion of small epitopes can change the heat modifiability of the OmpA TM domain from ‘aberrant’ to normal, and predict that some β-barrels will not display any significant heat-modifiability at all.
Chlamydia trachomatis ompA Variants in Trachoma: What Do They Tell Us?  [PDF]
Aura A. Andreasen,Matthew J. Burton,Martin J. Holland,Spencer Polley,Nkoyo Faal,David C.W. Mabey,Robin L. Bailey
PLOS Neglected Tropical Diseases , 2008, DOI: 10.1371/journal.pntd.0000306
Abstract: Background Trachoma, caused by Chlamydia trachomatis (Ct), is the leading infectious cause of blindness. Sequence-based analysis of the multiple strains typically present in endemic communities may be informative for epidemiology, transmission, response to treatment, and understanding the host response. Methods Conjunctival and nasal samples from a Gambian community were evaluated before and 2 months after mass azithromycin treatment. Samples were tested for Ct by Amplicor, with infection load determined by quantitative PCR (qPCR). ompA sequences were determined and their diversity analysed using frequency-based tests of neutrality. Results Ninety-five of 1,319 (7.2%) individuals from 14 villages were infected with Ct at baseline. Two genovars (A and B) and 10 distinct ompA genotypes were detected. Two genovar A variants (A1 and A2) accounted for most infections. There was an excess of rare ompA mutations, not sustained in the population. Post-treatment, 76 (5.7%) individuals had Ct infection with only three ompA genotypes present. In 12 of 14 villages, infection had cleared, while in two it increased, probably due to mass migration. Infection qPCR loads associated with infection were significantly greater for A1 than for A2. Seven individuals had concurrent ocular and nasal infection, with divergent genotypes in five. Conclusions The number of strains was substantially reduced after mass treatment. One common strain was associated with higher infection loads. Discordant genotypes in concurrent infection may indicate distinct infections at ocular and nasal sites. Population genetic analysis suggests the fleeting appearance of rare multiple ompA variants represents purifying selection rather than escape variants from immune pressure. Genotyping systems accessing extra-ompA variation may be more informative.
Bacillus subtilis SepF Binds to the C-Terminus of FtsZ  [PDF]
Ewa Król, Sebastiaan P. van Kessel, Laura S. van Bezouwen, Neeraj Kumar, Egbert J. Boekema, Dirk-Jan Scheffers
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0043293
Abstract: Bacterial cell division is mediated by a multi-protein machine known as the “divisome”, which assembles at the site of cell division. Formation of the divisome starts with the polymerization of the tubulin-like protein FtsZ into a ring, the Z-ring. Z-ring formation is under tight control to ensure bacteria divide at the right time and place. Several proteins bind to the Z-ring to mediate its membrane association and persistence throughout the division process. A conserved stretch of amino acids at the C-terminus of FtsZ appears to be involved in many interactions with other proteins. Here, we describe a novel pull-down assay to look for binding partners of the FtsZ C-terminus, using a HaloTag affinity tag fused to the C-terminal 69 amino acids of B. subtilis FtsZ. Using lysates of Escherichia coli overexpressing several B. subtilis cell division proteins as prey we show that the FtsZ C-terminus specifically pulls down SepF, but not EzrA or MinC, and that the interaction depends on a conserved 16 amino acid stretch at the extreme C-terminus. In a reverse pull-down SepF binds to full-length FtsZ but not to a FtsZΔC16 truncate or FtsZ with a mutation of a conserved proline in the C-terminus. We show that the FtsZ C-terminus is required for the formation of tubules from FtsZ polymers by SepF rings. An alanine-scan of the conserved 16 amino acid stretch shows that many mutations affect SepF binding. Combined with the observation that SepF also interacts with the C-terminus of E. coli FtsZ, which is not an in vivo binding partner, we propose that the secondary and tertiary structure of the FtsZ C-terminus, rather than specific amino acids, are recognized by SepF.
Characterization of Novel OmpA-Like Protein of Leptospira interrogans That Binds Extracellular Matrix Molecules and Plasminogen  [PDF]
Rosane Oliveira,Zenaide Maria de Morais,Amane Paldes Gon?ales,Eliete Caló Romero,Silvio Arruda Vasconcellos,Ana L. T. O. Nascimento
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0021962
Abstract: Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (KD, 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a KD of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.
Total Synthesis of Septocylindrin B and C-Terminus Modified Analogues  [PDF]
Jo Nelissen, Koen Nuyts, Marta De Zotti, Rob Lavigne, Chris Lamberigts, Wim M. De Borggraeve
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0051708
Abstract: The total synthesis is reported of the peptaibol Septocylindrin B which is related to the well documented channel forming peptaibol antibiotic Alamethicin. Several analogues were synthesized with a modified C-terminus, to investigate the SAR of the terminal residue Phaol. All these peptides were tested for their membrane perturbation properties by fluorescent dye leakage assay and for their antibacterial activity.
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