Presence Of Outer Membrane Proteins Research Articles

Antibiotic Resistance Profile, Outer Membrane Proteins, Virulence Factors and Genome Sequence Analysis Reveal Clinical Isolates of Enterobacter Are Potential Pathogens Compared to Environmental Isolates.

Outer membrane proteins (OMPs) of gram-negative bacteria play an important role in mediating antibacterial resistance, bacterial virulence and thus affect pathogenic ability of the bacteria. Over the years, prevalence of environmental antibiotic resistant organisms, their transmission to clinics and ability to transfer resistance genes, have been studied extensively. Nevertheless, how successful environmental bacteria can be in establishing as pathogenic bacteria under clinical setting, is less addressed. In the present study, we utilized an integrated approach of investigating the antibiotic resistance profile, presence of outer membrane proteins and virulence factors to understand extent of threat posed due to multidrug resistant environmental Enterobacter isolates. Also, we investigated clinical Enterobacter isolates and compared the results thereof. Results of the study showed that multidrug resistant environmental Enterobacter isolates lacked OmpC, lacked cell invasion abilities and exhibited low reactive oxygen species (ROS) production in neutrophils. In contrast, clinical isolates possessed OmpF, exhibited high invasive and adhesive property and produced higher amounts of ROS in neutrophils. These attributes indicated limited pathogenic potential of environmental Enterobacter isolates. Informations obtained from whole genome sequence of two representative bacterial isolates from environment (DL4.3) and clinical sources (EspIMS6) corroborated well with the observed results. Findings of the present study are significant as it highlights limited fitness of multidrug resistant environmental Enterobacter isolates.

Structures of Unfolded Outer Membrane Proteins in Complex with Chaperones

The viability of Gram-negative bacteria is ensured by the presence of outer membrane proteins (OMPs) acting as channels, porins, lipases and receptors. They are considered as interesting drug targets and thus obtaining a molecular perspective about various stages of their biogenesis is of great interest. However, the biogenesis of OMPs is biologically puzzling because it is not driven by ATP. We lack a detailed understanding of the various steps involved in this process. Unlike other proteins, OMPs adopt an active conformation only on reaching the outer membrane and thus have to be transferred from the cytoplasm through the periplasm in a folding competent state. Chaperones like Seventeen kilodalton protein (Skp) and Survival Factor A (SurA) protect these nascent hydrophobic proteins against aggregation. Yet, it remains elusive which conformations and interaction dynamics occur in the chaperone and unstructured OMP interplay. Using single molecule Förster Resonance Energy Transfer (smFRET), we investigate the structure of unfolded OMPs in chaperone-bound and unbound states. We find that both OMPs adopt a rather compact conformation in a low denaturant buffer. Remarkably, when bound with periplasmic chaperones, we observe that this compact state is dissolved by the chaperones differently for both OMPs. The small 8 beta-stranded OmpX shows heterogeneous conformations with sub-millisecond dynamics, while the 12 beta-stranded Outer membrane phospholipase A (OmpLA) appears to be pulled and pushed upon by the chaperones in expanded conformations. In both the cases, the chaperone-OMP interactions were remarkably long-lasting for hours, even below the dissociation constant. Our findings hint towards mechanisms by which OMPs are arranged in a folding competent state before reaching the outer membrane and shed light on the elusive mechanism of chaperone-OMP interaction in periplasm.

Characterization of the first planctomycetal outer membrane protein identifies a channel in the outer membrane of the anammox bacterium Kuenenia stuttgartiensis

Planctomycetes are a bacterial phylum known for their complex intracellular compartmentalization. While most Planctomycetes have two compartments, the anaerobic ammonium oxidizing (anammox) bacteria contain three membrane-enclosed compartments. In contrast to a long-standing consensus, recent insights suggested the outermost Planctomycete membrane to be similar to a Gram-negative outer membrane (OM). One characteristic component that differentiates OMs from cytoplasmic membranes (CMs) is the presence of outer membrane proteins (OMPs) featuring a β-barrel structure that facilitates passage of molecules through the OM. Although proteomic and genomic evidence suggested the presence of OMPs in several Planctomycetes, no experimental verification existed of the pore-forming function and localization of these proteins in the outermost membrane of these exceptional microorganisms. Here, we show via lipid bilayer assays that at least two typical OMP-like channel-forming proteins are present in membrane preparations of the anammox bacterium Kuenenia stuttgartiensis. One of these channel-forming proteins, the highly abundant putative OMP Kustd1878, was purified to homogeneity. Analysis of the channel characteristics via lipid bilayer assays showed that Kustd1878 forms a moderately cation-selective channel with a high current noise and an average single-channel conductance of about 170–190pS in 1M KCl. Antibodies were raised against the purified protein and immunogold localization indicated Kustd1878 to be present in the outermost membrane. Therefore, this work clearly demonstrates the presence of OMPs in anammox Planctomycetes and thus firmly adds to the emerging view that Planctomycetes have a Gram-negative cell envelope.

Label-free Gram-negative bacteria detection using bacteriophage-adhesin-coated long-period gratings.

This paper presents a novel application of a highly sensitive sensor based on long-period gratings (LPGs) coated with T4 bacteriophage adhesin for Gram-negative bacteria detection. We show here, that the sensor evidently recognizes Escherichia coli K-12 (PCM2560), whereas in the reference tests - ELISA and BIAcore - the results are questionable. For LPGs sensor the resonant wavelength shift observed for E. coli K-12 was approximately half of that measured for E.coli B (positive control). The BIAcore readings (RU) for E. coli K-12 were at 10% level of the signal obtained for E .coli B. These results confirm the improved sensitivity of the LPGs sensor. Moreover, we also show that application of adhesin may allow for efficient detection of E. coli O111 (PCM418), Klebsiella pneumoniae O1 (PCM1) and Yersinia enterocolitica O1 (PCM1879). The specificity of binding bacteria by the adhesin is discussed and it is determined by a distinct region of lipopolysaccharide receptors and/or by the presence of outer-membrane protein C in an outer membrane of Gram-negative bacteria.

Presumable role of outer membrane proteins of Salmonella containing sialylated lipopolysaccharides serovar Ngozi, sv. Isaszeg and subspecies arizonae in determining susceptibility to human serum.

BackgroundThe O48 group comprises Salmonella bacteria containing sialic acid in the lipopolysaccharide (LPS). Bacteria with sialylated surface structures are described as pathogens that avoid immunological response of the host by making similar their surface antigens to the host’s tissues (molecular mimicry). It is known that the smooth-type LPS of Salmonella enterica and outer membrane proteins (OMP) PgtE, PagC and Rck mediate serum resistant phenotype by affecting complement system (C). The aim of this study was to investigate C3 component activation by Salmonella O48 LPS and OMP.FindingsIn the present study, we examined C3 component deposition on the three Salmonella O48 strains: S. enterica subspecies enterica serovar Ngozi, S. enterica subsp. enterica sv. Isaszeg, and S.enterica subsp. arizonae containing sialic acid in the O-specific part of LPS. The greatest C3 deposition occurred on Salmonella sv. Isaszeg cells (p < 0.005) as well as on their LPS (low content of sialic acid in LPS) (p < 0.05) after 45 min of incubation in 50% human serum. Weaker C3 deposition ratio on the Salmonella sv. Ngozi (high content of sialic acid in LPS) and Salmonella subsp. arizonae (high content of sialic acid in LPS) cells correlated with the lower C3 activation on their LPS. Immunoblotting revealed that OMP isolated from the tested strains also bound C3 protein fragments.ConclusionsWe suggest that activation of C3 serum protein is dependent on the sialic acid contents in the LPS as well as on the presence of OMP in the range of molecular masses of 35–48 kDa.

Cellular, particle and environmental parameters influencing attachment in surface waters: a review

Effective modelling of the fate and transport of water-borne pathogens is needed to support federally required pollution-reduction plans, for water quality improvement planning, and to protect public health. Lack of understanding of microbial-particle interactions in water bodies has sometimes led to the assumption that bacteria move in surface waters not associated with suspended mineral and organic particles, despite a growing body of evidence suggesting otherwise. Limited information exists regarding the factors driving interactions between micro-organisms and particles in surface waters. This review discusses cellular, particle and environmental factors potentially influencing interactions and in-stream transport. Bacterial attachment in the aquatic environment can be influenced by properties of the cell such as genetic predisposition and physiological state, surface structures such as flagella and fimbriae, the hydrophobicity and electrostatic charge of the cell surface, and the presence of outer-membrane proteins and extracellular polymeric substances. The mechanisms and degree of attachment are also affected by characteristics of mineral and organic particles including the size, surface area, charge and hydrophobicity. Environmental conditions such as the solution chemistry and temperature are also known to play an important role. Just as the size and surface of chemical particles can be highly variable, bacterial attachment mechanisms are also diverse.

Assessment of extended-spectrum β-lactamase, KPC carbapenemase and porin resistance mechanisms in clinical samples of Klebsiella pneumoniae and Enterobacter spp.

The emergence and spread of resistance mechanisms in Gram-negative bacilli has complicated the treatment of serious nosocomial infections. Current automated systems for detection of Klebsiella pneumoniae carbapenemase (KPC)-producing isolates are unreliable. One possible straightforward alternative method is evaluation of ertapenem resistance. However, the accuracy of this method is affected by other resistance mechanisms such as AmpC gene expression or extended-spectrum β-lactamase production associated with porin loss. This study included 128 samples of K. pneumoniae and Enterobacter spp. that were non-susceptible to ertapenem. The disk diffusion and Etest method were applied to determine susceptibility to imipenem, meropenem and ertapenem. Isolates exhibiting intermediate or complete resistance to ertapenem were evaluated for resistance mechanisms. blaTEM, blaSHV, blaCTX-M, blaCTX-M-2 and blaKPC genes were tested for by PCR, and the presence of outer membrane protein was investigated by dot-blot assay. blaTEM was detected in 52.9% and 10.3%, blaSHV in 29.4% and 0.94%, blaCTX-M in 41.4% and 1.9% and blaCTX-M-2 in 23.5% and 1.9% of K. pneumoniae and Enterobacter cloacae isolates, respectively. The blaKPC gene was present in 12.6% of Enterobacter spp. isolates. OmpC and OmpF were present in 6.6% of E. cloacae isolates. These results indicate that several resistance mechanisms contribute to potential therapeutic failure of carbapenem therapy and point to the need for better detection methods and surveillance strategies.

High prevalence of OXA-143 and alteration of outer membrane proteins in carbapenem-resistant Acinetobacter spp. isolates in Brazil

Carbapenem resistance amongst Acinetobacter spp. has been increasing in the last decade. This study evaluated the outer membrane protein (OMP) profile and production of carbapenemases in 50 carbapenem-resistant Acinetobacter spp. isolates from bloodstream infections. Isolates were identified by API20NE. Minimum inhibitory concentrations (MICs) for carbapenems were determined by broth microdilution. Carbapenemases were studied by phenotypic tests, detection of their encoding gene by polymerase chain reaction (PCR) amplification, and imipenem hydrolysis. Nucleotide sequencing confirming the enzyme gene type was performed using MegaBACE 1000. The presence of OMPs was studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and PCR. Molecular typing was performed using pulsed-field gel electrophoresis (PFGE). All isolates were resistant to carbapenems. Moreover, 98% of the isolates were positive for the gene encoding the enzyme OXA-51-like, 18% were positive for OXA-23-like (only one isolate did not show the presence of the insertion sequence ISAba1 adjacent to this gene) and 76% were positive for OXA-143 enzyme. Five isolates (10%) showed the presence of the IMP-1 gene. Imipenem hydrolysing activity was detected in only three strains containing carbapenemase genes, comprising two isolates containing the blaIMP gene and one containing the blaOXA-51/OXA-23-like gene. The OMP of 43kDa was altered in 17 of 25 strains studied, and this alteration was associated with a high meropenem MIC (256μg/mL) in 5 of 7 strains without 43kDa OMP. On the other hand, decreased OMP 33–36kDa was found in five strains. The high prevalence of OXA-143 and alteration of OMPs might have been associated with a high level of carbapenem resistance.

Characterization of outer membrane protein fractions of Bdellovibrionales

Bdellovibrio-and-like organisms (BALOs) are predatory bacteria that prey upon Gram-negative bacteria and are taxonomically subsumed in the order Bdellovibrionales. Despite their unique lifestyle, these bacteria show remarkable genotypic diversities. The outer membrane of the predators is likely to play an important role during the recognition and invasion stage, as well as in the intraperiplasmic growth phase. In this study, the outer membrane protein fractions of type strains of Bdellovibrio, Bacteriovorax and Peredibacter were investigated, revealing the presence of outer membrane proteins (Omps) similar to the major Omps of Bdellovibrio bacteriovorus. The primary structures of these Omps of Bdellovibrio sp. W, Bacteriovorax stolpii and Peredibacter starrii were elucidated by a combined mass spectrometric–reverse genetic approach. The similarity between the analyzed Omps of the investigated BALOs ranges from 32% to 89% showing conserved amino acid regions in their primary structure.

In vitro degradation and release profiles of poly-DL-lactide-poly(ethylene glycol) microspheres with entrapped proteins

Poly-DL-lactide (PLA) and poly-DL-lactide-poly(ethylene glycol) (PELA) were produced by bulk ring-opening polymerization using stannous chloride as initiator. PLA, PELA microspheres, and PELA microspheres containing the outer membrane protein (OMP) of Leptospira interrogans with the size of 1.5–2 μm were prepared by a solvent evaporation process. In vitro degradation and release tests of PLA, PELA, and OMP-loaded PELA microspheres were performed in pH 7.4 buffer solution at 37°C. Quantitatively, the degree of degradation was monitored by detecting the molecular weight reduction, by evaluating the mass loss and the apparent degradation rate constant, and by determining the intrinsic viscosity and poly(ethylene glycol) content of retrieved polymer, while the release profile was assessed by measuring the amount of protein presented in the release medium at various intervals. Qualitatively, the morphological changes of microspheres were observed with scanning electron micrography. The observed relative rates of mass loss versus molecular weight reduction are consistent with a bulk erosion process rather than surface erosion for PELA microspheres. The introduction of hydrophilic poly(ethylene glycol) domains in copolymer PELA and the presence of OMP within microspheres show critical influences on the degradation profile. The OMP-loaded PELA microspheres present triphasic release profile and a close correlation is observed between the polymer degradation and the OMP release profiles. It is suggested that the polymer degradation rate, protein diffusion coefficient, and the water-swollen structure of microspheres matrix commonly contribute to the OMP release from PELA microspheres. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 78: 140–148, 2000

Characterization of inclusion bodies in recombinant Escherichia coli producing high levels of porcine somatotropin

The protein composition of inclusion bodies (IBs) formed in recombinant Escherichia coli producing high levels of porcine somatotropin (pST) was analyzed by one- and two-dimensional protein gel electrophoresis. Recombinant pST is exclusively recovered from the insoluble cell fraction. Results indicate that, in addition to the main species of pST, subspecies with different isoelectric points and degradative fragments are contained within IBs. The presence of outer membrane proteins in IB fractions results from coprecipitation of cell debris during IB preparation and not from specific in vivo or in vitro interaction of these proteins with IBs. Cells producing pST contain up to three IBs located in the cytoplasm. The implication of high level gene expression on the uniformity of the desired product is discussed.

Colony opacity and protein II compositions of gonococci.

Changes in the presence of outer membrane protein II (P.II) constituents of gonococci were demonstrated by selecting opacity variants and defining the 125I-labeled bands of parental and variant organisms. In general, colony opacity phenotype was a convenient, reliable guide for obtaining variants that differed from their parents by the apparent single-step gain or loss of one P.II constituent. Within a given strain (three strains were studied), particular P.II species were associated with particular opacity phenotypes. This was well demonstrated in strain JS3, in which five different P.II constituents were identified and compared. Four of these five P.II moieties were consistently associated with a characteristic degree of colony opacity: presence of the fifth P.II (P.IIa) did not correlate with a discernible increase in opacity when present either alone or in combination with other opacity-associated P.II moieties. The electrophoretic migration characteristics for each of the five P.II constituents of this strain differed with regard to apparent molecular weight and the effects of temperature and 2-mercaptoethanol. The high prevalence of colony opacity variants indicates that gonococcal populations are capable of presenting a variety of surface components to their external environment.

Reconstitution of Model Membranes from Phospholipid and Outer Membrane Proteins of Proteus mirabilis

Outer membrane proteins extracted from isolated cell walls of Proteus mirabilis were able to combine the cell wall phospholipids in a model membrane system. The presence of outer membrane proteins in vesicular model membranes mediated the release of previously entrapped [14C]sucrose while [3H]inulin was retained. Incorporation of lipopolysaccharide from the same cell walls was not required for the formation of such selectively permeable membranes. Three major outer membrane proteins of apparent molecular weights 39000, 36000 and 17000 were isolated using acetic acid and sodium deoxycholate solution as solvents and avoiding the strongly denaturing sodium dodecyl sulfate. The isolated proteins were assayed for their ability to form hydrophilic pores in reconstituted membranes. The trypsin-sensitive 39000-Mr protein and the peptidoglycan-associated 36000-Mr protein were equally effective in this function whereas the 17000-Mr protein mediated little penetration of low molecular weight solute. The 39000-Mr and 36000-Mr proteins also protected reconstituted membrane vesicles from disruption by detergent while 17000-Mr protein was ineffective in this regard.