One-Step Purification and Porin Transport Activity of the Major Outer Membrane Proteins P2 from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis

Kattner, Christof; Pfennig, Sabrina; Massi, Paola; Tanabe, Mikio

doi:10.1007/s12010-014-1473-2

Cited by 5 publications

(9 citation statements)

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“…Isolate 2014-5 showed a high MIC value (0.5 µg/ml) for not only MXF but also CIP and LVX, although this isolate had no mutations in QRDRs of both GyrA and ParC enzymes (Table 1). A factor presumably involved in this is a drug efflux pump (acrAB) (21–22) or H. influenzae -specific porin (23–24), the protein structure of which is similar to that of porin (OmpF); however, solid evidence was not obtained from the present study, and this point remains to be addressed in future studies.…”

Section: Discussionmentioning

confidence: 60%

Monitoring quinolone resistance due to Haemophilus influenzae mutations (2012–17)

Nagatomo

Shirakura

Fukuchi

et al. 2019

Preprint

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17Among drug-resistant bacteria of recent concern, we determined minimum inhibitory 18 concentrations (MICs) of six different quinolone antibacterial agents in Haemophilus 19 influenzae and performed molecular genetic analysis in addition to the exploration for 20 β-lactamase-producing and β-lactamase negative ampicillin-resistant H. influenzae 21 (BLNAR). A total of 144 clinical H. influenzae strains isolated at the Showa University 22 Hospital between 2012 and 2017 were subjected to MIC determination for 23 penicillin/quinolone antibacterial agents using the nitrocefin method and the Clinical 24 and Laboratory Standards Institute broth microdilution method. Moreover, amino acid 25 mutations in the quinolone resistance-determining regions (QRDRs) were analyzed in 26 the isolates showing MIC value of ≥ 0.25 µg/ml of quinolone antibacterial agents. 27Increasing proportions of BLNAR were noted, with 15% in 2015 to 43.5% in 2016 and 28 63.6% in 2017. Among quinolone antibacterial agents, all isolates remained susceptible 29 to sitafloxacin (STFX), and STFX showed strong inhibitory potencies against both 30 DNA gyrase and topoisomerase IV. For moxifloxacin (MXF), however, strains with 31 MIC value of 0.5 µg/ml were detected every year since 2013 except 2015. Amino acid 32 mutations were investigated in 17 isolates (11.8%) with MXF MIC value of ≥0.25 33 µg/ml, and confirmed in 11 isolates (7.6%), of which mutations of GyrA were found in 34 3 3 9 isolates. Future antibacterial drug regimens may need to address the emergence of 35 quinolone-resistant H. influenzae. 36 37 38 4 4 Introduction 39 Haemophilus influenzae is the second most common causative bacterium of 40 community-acquired pneumonia, following Streptococcus pneumoniae, and has taken 41 the leading position after pneumococcal vaccines became popular (1). β-Lactam 42 antibiotics have previously been used for the treatment of community-acquired 43 pneumonia. In the 2000s, however, β-lactamase producing ampicillin-resistant (BLPAR) 44 bacteria have been increasingly detected in Canada (2), Japan, and some other countries. 45 In addition, β-lactamase negative ampicillin-resistant (BLNAR) bacteria have been 46 increasingly detected in Japan in the recent years (3). Moreover, BLNAR bacteria 47 65 GyrA gene encoding DNA gyrase or ParC gene encoding topoisomerase IV, which 66 cause three-dimensional structural alterations in the respective enzymes and decrease 67 the affinity between fluoroquinolones and these replication enzymes (9). In the case of 68 H. influenzae, drug resistance has been shown to be strongly related to mutations of 69 serine and aspartic acid at positions 84 and 88 of GyrA and of serine at position 84 of 70 ParC (10). However, only a limited number of previous studies have investigated 71 mutations in QRDRs in the same institution over time or have compared MICs among 72 different quinolones.In this study, we tested sensitivity levels to quinolone and 73 penicillin antibacterial agents over 6 years in H. influenzae strains that were isolated at ...

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Section: Discussionmentioning

confidence: 60%

Monitoring quinolone resistance due to Haemophilus influenzae mutations (2012–17)

Nagatomo

Shirakura

Fukuchi

et al. 2019

Preprint

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“…Branching lower within cluster X is the OmpG OMPP (OmpG) Family (1.B.21), and closest to the center of the tree, we find the Fusobacterial Outer Membrane OMPP (FomA) Family (1.B.32). Proteins of these two families have about the same sizes and numbers of β-TMSs (14) as the CDP and KdgM families, but they are reported to catalyze non-specific export of small molecules, restricted only by the sizes of the substrates [ 110 – 112 ].…”

Section: Resultsmentioning

confidence: 99%

Properties and Phylogeny of 76 Families of Bacterial and Eukaryotic Organellar Outer Membrane Pore-Forming Proteins

Reddy

Saier

2016

PLoS ONE

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We here report statistical analyses of 76 families of integral outer membrane pore-forming proteins (OMPPs) found in bacteria and eukaryotic organelles. 47 of these families fall into one superfamily (SFI) which segregate into fifteen phylogenetic clusters. Families with members of the same protein size, topology and substrate specificities often cluster together. Virtually all OMPP families include only proteins that form transmembrane pores. Nine such families, all of which cluster together in the SFI phylogenetic tree, contain both α- and β-structures, are multi domain, multi subunit systems, and transport macromolecules. Most other SFI OMPPs transport small molecules. SFII and SFV homologues derive from Actinobacteria while SFIII and SFIV proteins derive from chloroplasts. Three families of actinobacterial OMPPs and two families of eukaryotic OMPPs apparently consist primarily of α-helices (α-TMSs). Of the 71 families of (putative) β-barrel OMPPs, only twenty could not be assigned to a superfamily, and these derived primarily from Actinobacteria (1), chloroplasts (1), spirochaetes (8), and proteobacteria (10). Proteins were identified in which two or three full length OMPPs are fused together. Family characteristic are described and evidence agrees with a previous proposal suggesting that many arose by adjacent β-hairpin structural unit duplications.

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“…PorB G103K from Neisseria meningitidis W135 strains was expressed, purified and crystallized as described previously [17, 23, 42, 43]. Crystals of PorB in the P6 3 crystal form were obtained in 100 mM MES, pH 6.5, 31% Jeffamine M-600.…”

Section: Methodsmentioning

confidence: 99%

“…To test for ampicillin transport activity of PorB wt and PorB G103K, a liposome swelling assay was performed at isoosmotic conditions as described previously [42]. Multilamellar liposomes were prepared using E. coli polar lipid extract (Avanti Polar Lipid, Alabaster) [21, 23].…”

Section: Methodsmentioning

confidence: 99%

An antibiotic-resistance conferring mutation in a neisserial porin: Structure, ion flux, and ampicillin binding

Bartsch

Ives

Kattner

et al. 2020

Preprint

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Gram-negative bacteria cause the majority of highly drug-resistant bacterial infections. To cross the outer membrane of the complex Gram-negative cell envelope, antibiotics permeate through porins, trimeric channel proteins that enable the exchange of small polar molecules. Mutations in porins contribute to the development of drug-resistant phenotypes. In this work, we show that a single point mutation in the porin PorB from Neisseria meningitidis, the causative agent of bacterial meningitis, can strongly affect the binding and permeation of beta-lactam antibiotics. Using X-ray crystallography, high-resolution electrophysiology, atomistic biomolecular simulation, and liposome swelling experiments, we demonstrate differences in drug binding affinity, ion selectivity and drug permeability of PorB. Our work further reveals distinct interactions between the transversal electric field in the porin eyelet and the zwitterionic drugs, which manifest themselves under applied electric fields in electrophysiology and are altered by the mutation. These observations may apply more broadly to drug-porin interactions in other channels. Our results improve the molecular understanding of porin-based drug-resistance in Gram-negative bacteria.

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One-Step Purification and Porin Transport Activity of the Major Outer Membrane Proteins P2 from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis

Cited by 5 publications

References 32 publications

Monitoring quinolone resistance due to Haemophilus influenzae mutations (2012–17)

Monitoring quinolone resistance due to Haemophilus influenzae mutations (2012–17)

Properties and Phylogeny of 76 Families of Bacterial and Eukaryotic Organellar Outer Membrane Pore-Forming Proteins

An antibiotic-resistance conferring mutation in a neisserial porin: Structure, ion flux, and ampicillin binding

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