Synchrotron FTIR and Raman spectroscopy provide unique spectral fingerprints for Arabidopsis floral stem vascular tissues.

Cell walls are highly complex structures that are modified during plant growth and development. For example, the development of phloem and xylem vascular cells, which participate in the transport of sugars and water as well as providing support, can be influenced by cell-specific wall composition. Here, we used synchrotron radiation-based Fourier-transform infrared (SR-FTIR) and Raman spectroscopy to analyse the cell wall composition of floral stem vascular tissues of wild-type Arabidopsis and the double-mutant sweet11-1 sweet12-1, which has impaired sugar transport. The SR-FTIR spectra showed that in addition to modified xylem cell wall composition, phloem cell walls in the double-mutant line were characterized by modified hemicellulose composition. Combining Raman spectroscopy with a classification and regression tree (CART) method identified combinations of Raman shifts that could distinguish xylem vessels and fibers. In addition, the disruption of the SWEET11 and SWEET12 genes impacted on xylem wall composition in a cell-specific manner, with changes in hemicelluloses and cellulose observed at the xylem vessel interface. These results suggest that the facilitated transport of sugars by transporters that exist between vascular parenchyma cells and conducting cells is important in ensuring correct phloem and xylem cell wall composition.

Understanding the responses of Saccharomyces cerevisiae yeast strain during dehydration processes using synchrotron infrared spectroscopy.

For the first time, synchrotron infrared spectroscopy was performed on yeast during dehydration processes in real time with simultaneously controlled relative humidity and temperature. This led us to investigate the biochemical modification in relation to the dehydration of Saccharomyces cerevisiae. The correlation between the hydration level and yeast survival was observed. Following the test conditions, the modification of the protein structure was observed. However, no evident modification of the lipid composition resulting from dehydration was observed. Furthermore, the results showed that the medium rich in nutrients and glutathione precursors can improve yeast survival during dehydration at 45 °C. This could be related to the high relative amounts of CH3 groups in the lipid composition assigned to the low lipid oxidation level in this case. Our work demonstrated the feasibility of using S-FTIR for investigating yeast responses to dehydration processes in real time. This method can be used for understanding the effect of dehydration/rehydration on the biochemical modification of yeast.

A compensatory mutagenesis study of a conserved hairpin in the M gene segment of influenza A virus shows its role in virus replication.

RNA structures are increasingly recognized to be of importance during influenza A virus replication. Here, we investigated a predicted conserved hairpin in the M gene segment (nt 967-994) within the region of the vRNA 5' packaging signal. The existence of this RNA structure and its possible role in virus replication was investigated using a compensatory mutagenesis approach. Mutations were introduced in the hairpin stem, based on natural variation. Virus replication properties were studied for the mutant viruses with disrupted and restored RNA structures. Viruses with structure-disrupting mutations had lower virus titers and a significantly reduced median plaque size when compared with the wild-type (WT) virus, while viruses with structure restoring-mutations replicated comparable to WT. Moreover, virus replication was also reduced when mutations were introduced in the hairpin loop, suggesting its involvement in RNA interactions. Northern blot and FACS experiments were performed to study differences in RNA levels as well as production of M1 and M2 proteins, expressed via alternative splicing. Stem-disruptive mutants caused lower vRNA and M2 mRNA levels and reduced M2 protein production at early time-points. When the RNA structure was restored, vRNA, M2 mRNA and M2 protein levels were increased, demonstrating a compensatory effect. Thus, this study provides evidence for functional importance of the predicted M RNA structure and suggests its role in splicing regulation.

Unsupervised explorative data analysis of normal human leukocytes and BCR/ABL positive leukemic cells mid-infrared spectra.

We proved the ability of Fourier Transform Infrared microspectroscopy (microFTIR) complemented by Principal Component Analysis (PCA) to detect protein phosphorylation/de-phosphorylation in mammalian cells. We analyzed by microFTIR human polymorphonuclear neutrophil (PMNs) leukocytes, mouse-derived parental Ba/F3 cells (Ba/F3#PAR), Ba/F3 cells transfected with p210(BCR/ABL) (Ba/F3#WT) and expressing high levels of protein tyrosine kinase (PTK), and human-derived BCR/ABL positive K562 leukemic cell sub-clones engineered to differently express receptor-type tyrosine-protein phosphatase gamma (PTPRG). Synchrotron radiation (SR) and conventional (globar) IR sources were used to perform microFTIR respectively, on single cells and over several cells within the same sample. Ex vivo time-course experiments were run, inducing maximal protein phosphorylation in PMNs by 100 nM N-formylated tripeptide fMLP. Within the specific IR fingerprint 1800-850 cm(-1) frequency domain, PCA identified two regions with maximal signal variance. These were used to model and test the robustness of PCA in representing the dynamics of protein phosphorylation/de-phosphorylation processes. An IR signal ratio marker reflecting the homeostatic control by protein kinases and phosphatases was identified in normal leukocytes. The models identified by microFTIR and PCA in normal leukocytes also distinguished BCR/ABL positive Ba/F3#WT from BCR/ABL negative Ba/F3#PAR cells as well as K562 cells exposed to functionally active protein tyrosine phosphatase recombinant protein ICD-Tat transduced in cells by HIV-1 Tat technology or cells treated with the PTK inhibitor imatinib mesylate (IMA) from cells exposed to phosphatase inactive (D1028A)ICD-Tat recombinant protein and untreated control cells, respectively. The IR signal marker correctly reflected the degrees of protein phosphorylation associated with abnormal PTK activity in BCR/ABL positive leukemic cells and in general was inversely related to the expression/activity of PTPRG in leukemic sub-clones. In conclusion, we have described a new, reliable and simple spectroscopic method to study the ex vivo protein phosphorylation/de-phosphorylation balance in cell models: it is suitable for biomedical and pharmacological research labs but it also needs further optimization and its evaluation on large cohorts of patients to be proposed in the clinical setting of leukemia.

Differences in the hippocampal frequency of creatine inclusions between the acute and latent phases of pilocarpine model defined using synchrotron radiation-based FTIR microspectroscopy.

Temporal lobe epilepsy (TLE) is the most common type of epilepsy in adults. Of the animal models developed to investigate the pathogenesis of TLE, the one with pilocarpine-induced seizures is most often used. After pilocarpine administration in animals, three distinct periods--acute, latent, and chronic--can be distinguished according to their behavior. The present paper is the continuation of our previous study which has shown an increased occurrence of creatine inclusions in rat hippocampal formations from the acute phase of pilocarpine-induced status epilepticus (SE) and positive correlation between their quantity and the total time of seizure activity within the observation period. In this paper, we tried to verify if anomalies in hippocampal creatine accumulation were the temporary or permanent effect of pilocarpine-evoked seizures. To realize this purpose, male Wistar rats in the latent phase (3 days after pilocarpine administration) were examined. The results obtained for the period when stabilization of animal behavior and EEG occurs were afterwards compared with ones obtained for the acute phase of pilocarpine-induced SE and for naive controls. To investigate the frequency of creatine inclusions within the hippocampal formation as well as in its selected areas (sectors 1-3 of Ammon's horn (CA1-CA3), dentate gyrus (DG), and hilus of DG) and cellular layers (pyramidal, molecular, multiform, and granular cell layers), synchrotron radiation-based Fourier-transform infrared microspectroscopy was used. The applied technique, being a combination of light microscopy and infrared spectroscopy, allowed us to localize microscopic details in the analyzed samples and provided information concerning their chemical composition. Moreover, the use of a synchrotron source of IR radiation allowed us to carry out the research at the diffraction-limited spatial resolution which, because of the typical size of creatine inclusions (from a few to dozens of micrometers), was necessary for our study. The comparison of epileptic animals in the latent phase with controls showed statistically significant increase in the number of creatine inclusions for most of the analyzed hippocampal regions, all examined cellular layers, as well as the whole hippocampal formation. Moreover, for the hilus of the DG and CA3 area, the number of creatine deposits was higher in the latent than in the acute phase after pilocarpine injection. In light of the obtained results, an anomaly in the hippocampal accumulation of creatine is the long-term effect of pilocarpine-evoked seizures, and the intensity of this phenomenon may increase with time passing from the primary injury.

Synchrotron radiation Fourier-transform infrared and Raman microspectroscopy study showing an increased frequency of creatine inclusions in the rat hippocampal formation following pilocarpine-induced seizures.

In the present work, synchrotron radiation Fourier-transform infrared (SRFTIR) and Raman microspectroscopies were used to evaluate a possible role of creatine in the pathogenesis and progress of pilocarpine-evoked seizures and seizure-induced neurodegenerative changes in the rat hippocampal tissue. The main goal of this study was to identify creatine deposits within the examined brain area, to analyze their frequency in epileptic animals and naive controls and to examine correlations between the number of inclusions in the hippocampal formation of epileptic rats and the quantitative parameters describing animal behavior during 6-h observation period after pilocarpine injection. The presence of creatine in the brain tissue was confirmed based on the vibrational bands specific for this compound in the infrared and Raman spectra. These were the bands occurring at the wavenumbers around 2800, 1621, 1398, and 1304 cm(-1) in IR spectra and around 1056, 908 and 834 cm(-1) in the Raman spectra. Creatine was detected in eight of ten analyzed epileptic samples and in only one of six controls under the study. The number of deposits in epileptic animals varied from 1 to 100 and a relative majority of inclusions were detected in the area of the Dentate Gyrus and in the multiform hippocampal layer. Moreover, the number of creatine inclusions was positively correlated with the total time of seizure activity.

Longitudinal humoral response after SARS-CoV-2 vaccination in ocrelizumab treated MS patients: To wait and repopulate?

OBJECTIVE: The objective of this study was to measure humoral responses after SARS-CoV-2 vaccination in MS patients treated with ocrelizumab (OCR) compared to MS patients without disease modifying therapies (DMTs) in relation to timing of vaccination and B-cell count. METHODS: OCR treated patients were divided into an early and a late group (cut-off time 12 weeks between infusion and first vaccination). Patients were vaccinated with mRNA-1273 (Moderna). B-cells were measured at baseline (time of first vaccination) and SARS-CoV-2 antibodies were measured at baseline, day 28, 42, 52 and 70. RESULTS: 87 patients were included (62 OCR patients, 29 patients without DMTs). At day 70, seroconversion occurred in 39.3% of OCR patients compared to 100% of MS patients without DMTs. In OCR patients, seroconversion varied between 26% (early group) to 50% (late group) and between 27% (low B-cells) to 56% (at least 1 detectable B-cell/µL). CONCLUSIONS: Low B-cell counts prior to vaccination and shorter time between OCR infusion and vaccination may negatively influence humoral response but does not preclude seroconversion. We advise OCR treated patients to get their first vaccination as soon as possible. In case of an additional booster vaccination, timing of vaccination based on B-cell count and time after last infusion may be considered.

Identification of distinct pathological signatures induced by patient-derived α-synuclein structures in nonhuman primates.

Dopaminergic neuronal cell death, associated with intracellular α-synuclein (α-syn)-rich protein aggregates [termed "Lewy bodies" (LBs)], is a well-established characteristic of Parkinson's disease (PD). Much evidence, accumulated from multiple experimental models, has suggested that α-syn plays a role in PD pathogenesis, not only as a trigger of pathology but also as a mediator of disease progression through pathological spreading. Here, we have used a machine learning-based approach to identify unique signatures of neurodegeneration in monkeys induced by distinct α-syn pathogenic structures derived from patients with PD. Unexpectedly, our results show that, in nonhuman primates, a small amount of singular α-syn aggregates is as toxic as larger amyloid fibrils present in the LBs, thus reinforcing the need for preclinical research in this species. Furthermore, our results provide evidence supporting the true multifactorial nature of PD, as multiple causes can induce a similar outcome regarding dopaminergic neurodegeneration.

Challenging immunodominance of influenza-specific CD8+ T cell responses restricted by the risk-associated HLA-A*68:01 allomorph.

Although influenza viruses lead to severe illness in high-risk populations, host genetic factors associated with severe disease are largely unknown. As the HLA-A*68:01 allele can be linked to severe pandemic 2009-H1N1 disease, we investigate a potential impairment of HLA-A*68:01-restricted CD8+ T cells to mount robust responses. We elucidate the HLA-A*68:01+CD8+ T cell response directed toward an extended influenza-derived nucleoprotein (NP) peptide and show that only ~35% individuals have immunodominant A68/NP145+CD8+ T cell responses. Dissecting A68/NP145+CD8+ T cells in low vs. medium/high responders reveals that high responding donors have A68/NP145+CD8+ memory T cells with clonally expanded TCRαßs, while low-responders display A68/NP145+CD8+ T cells with predominantly naïve phenotypes and non-expanded TCRαßs. Single-cell index sorting and TCRαß analyses link expansion of A68/NP145+CD8+ T cells to their memory potential. Our study demonstrates the immunodominance potential of influenza-specific CD8+ T cells presented by a risk HLA-A*68:01 molecule and advocates for priming CD8+ T cell compartments in HLA-A*68:01-expressing individuals for establishment of pre-existing protective memory T cell pools.

Quantification of local water and biomass in wild type PA01 biofilms by Confocal Raman Microspectroscopy.

Confocal Raman Microspectroscopy (CRM) can be used as a tool for the in situ evaluation of the chemical composition of living, fully submerged, unstained biofilms. In this study the estimation of the local water content in Pseudomonas aeruginosa PA01 biofilms is given as an example. The ratio of the area of the O-H stretching vibration band at 3450 cm(-1), (water), to that of the C-H stretching bands at 2950 cm(-1) (biomass), was used to estimate the relative biofilm water content. The quantification of biofilm water and biomass was based on calibration curves generated from protein solutions. Water/biomass ratios (W:BR) equivalent to that of a 30% (w/v) protein solution were observed within some biofilm colonies.

Photophysical properties of single core multimodal probe for imaging (SCoMPI) in a membrane model and in cells.

The spectroscopic properties of two luminescent Re(i) tricarbonyl complexes conjugated with two cell-penetrating peptides were examined. Fluorescence experiments and IR quantification in membrane models and in cells showed unexpectedly strong luminescence enhancement for one of the complexes in a lipid environment.

Prevalence and Spatial Distribution of Salmonella Infections in the Pennsylvania Raccoon (Procyon lotor).

A study was conducted to determine the prevalence and spatial distribution of Salmonella infection in Pennsylvania raccoons (Procyon lotor), common wildlife mammals known to occupy overlapping habitats with humans and domestic food animals. The Pennsylvania Game Commission provided a total of 371 raccoon intestinal samples from trapped and road-killed raccoons collected between May and November 2011. Salmonella was isolated from the faeces of 56 (15.1%) of 371 raccoons in 35 (54%) of 65 counties across Pennsylvania. The five most frequently isolated serotypes were Newport (28.6%), Enteritidis (19.6%), Typhimurium (10.7%), Braenderup (8.9%) and Bareilly (7.1%). Pulsed-field gel electrophoresis (PFGE) analysis of the Salmonella isolates and subsequent comparison to the Pennsylvania Department of Health human Salmonella PFGE database revealed 16 different pulsetypes in Salmonella isolates recovered from raccoons that were indistinguishable from pulsetypes of Salmonella collected from clinically ill humans during the study period. The pulsetypes of seven raccoon Salmonella isolates matched those of 56 human Salmonella isolates by month and geographical region of sample collection. Results from Clustered Regularly Interspaced Short Palindromic Repeats and Multi-Virulence Locus Sequence Typing (CRISPR-MVLST) analysis corroborated the PFGE and serotyping data. The findings of this study show that several PFGE pulsetypes of Salmonella were shared between humans and raccoons in Pennsylvania, indicating that raccoons and humans might share the same source of Salmonella.

Correlation of Rhs elements with Escherichia coli population structure.

The Rhs family of composite genetic elements was assessed for variation among independent Escherichia coli strains of the ECOR reference collection. The location and content of the RhsA-B-C-F subfamily correlates highly with the clonal structure of the ECOR collection. This correlation exists at several levels: the presence of Rhs core homology in the strain, the location of the Rhs elements present, and the identity of the Rhs core-extensions associated with each element. A provocative finding was that an identical 1518-bp segment, covering core-extension-b1 and its associated downstream open reading frame, is present in two distinct clonal groups, but in association with different Rhs elements. The sequence identity of this segment when contrasted with the divergence of other chromosomal segments suggests that shuffling of Rhs core extensions has been a relatively recent variation. Nevertheless the copies of core-extension-b1 were placed within the respective Rhs elements before the emergence of the clonal groups. In the course of this analysis, two new Rhs elements absent from E. coli K-12 were discovered: RhsF, a fourth member of the RhsA-B-C-F subfamily, and RhsG, the prototype of a third Rhs subfamily.

Analysis of structural changes in normal and aneurismal human aortic tissues using FTIR microscopy.

Aortic aneurisms are frequently asymptomatic but can induce dramatic complications. The diagnosis is only based on the aortic diameter and not on a structural and compositional basis. In this preliminary study, we propose infrared microspectroscopy to nondestructively probe normal and aneurismal human aortas. Spectra from 19 human ascending aortic biopsies (10 normal and 9 aneurismal) were acquired using infrared microspectroscopy. A 1500 x 150 microm(2) area of each 7-microm thick cryosection was investigated using a 30-microm spatial resolution with a total of about 200 spectra per sample. Spectral differences between normal and aneurismal tissues were mainly located in spectral regions related to proteins, such as elastin and collagen, and proteoglycans (1750-1000 cm(-1)). Tissue heterogeneity and sample classification have been evaluated using hierarchical cluster analysis of individual or mean spectra and their second derivative. Using spectral range related to proteins, 100% of good classification was obtained whereas the proteoglycan spectral range was less discriminant. This in vitro study demonstrates the potential of such technique to differentiate between normal and aneurismal aortas using selected spectral ranges. Future investigations will be focused on these specific spectral regions to determine the role of elastin and collagen in the discrimination of normal and pathological aortas.

Confocal Raman microspectroscopy as a tool for studying the chemical heterogeneities of biofilms in situ.

AIMS: To investigate the use of confocal Raman microspectroscopy (CRM) for the analysis of the structure, composition and development of fully hydrated biofilms. METHODS AND RESULTS: Pseudomonas aeruginosa PAO1 biofilms were cultured in a flow cell in minimal nutrient medium (artificial sea water) and their development was followed for up to 3 weeks. The spectroscopic signature of the biofilm cells and extracellular polymeric substances (EPS) were differentiated and their distribution in biofilm colonies and within water channels was mapped in-plane and -depth. The colonies were initially amorphous, mainly composed of cells with no detectable amount of EPS. They developed rapidly to give round colonies composed of a cellular core enclosed in a sheath of EPS. The EPS continued to increase and spread throughout the biofilm to become the dominating feature of aged colonies. Colonies with a liquid core morphology - characteristic of the seeding dispersal process - were also observed. CONCLUSIONS: This study demonstrated that CRM can be used to monitor the distribution of biofilm components in fully hydrated undisturbed biofilms over time. SIGNIFICANCE AND IMPACT OF THE STUDY: Confocal Raman microspectroscopy facilitates the analysis of hydrated, live bacterial biofilms as a function of space and time, thus making it a suitable technique for investigating the effects of various additives and environmental factors on biofilm growth.

Outbreak of Salmonella Braenderup infections associated with Roma tomatoes, northeastern United States, 2004: a useful method for subtyping exposures in field investigations.

Salmonella Braenderup is an uncommon serotype in the United States. In July 2004, a multistate outbreak of Salmonella Braenderup diarrhoeal infections occurred, with 125 clinical isolates identified. To investigate, we conducted a case-control study, enrolling 32 cases and 63 matched controls. Cheese, lettuce and tomato eaten at restaurants all appeared to be associated with illness. To further define specific exposures, we conducted a second study and asked managers of restaurants patronized by patients and controls about cheese, lettuce and tomato varieties used in dishes their patrons reported consuming. This information was obtained for 27 cases and 29 controls. Roma tomatoes were the only exposure significantly associated with illness (odds ratio 4.3, 95% confidence interval 1.2-15.9). Roma tomatoes from two restaurants were traced back to a single tomato packing house. The methods used in this field investigation to define specific exposures may be useful for other foodborne outbreaks.

FT-IR microspectroscopy for early identification of some clinically relevant pathogens.

AIMS: To investigate the potentials and limitations of Fourier transform-infrared (FT-IR) microspectroscopy as a tool to identify, at the level of microcolonies, pathogenic bacteria frequently isolated in the clinical environment. METHODS AND RESULTS: A total of 1570 FT-IR spectra from 164 gram-positive and gram-negative bacteria isolated from patients were recorded from 6 to 10-h old microcolonies of 50-150 microm size. A classification of 100% was obtained for the most frequent gram-positive bacteria, such as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium at the species level. An average accuracy of about 80% was reached with Gram negative bacteria from the Enterobacteriaceae and Pseudomonaceae families; Enterobacter aerogenes, Enterobacter cloacae, Klebsiella spp., and Citrobacter koseri; and Proteus mirabilis and Escherichia coli. Results were comparable with FT-IR measurements on dried suspensions from 18-h cultures. CONCLUSIONS: Early identification of young microcolonies is feasible with FT-IR microscopy with a very high accuracy for gram-positive bacteria. Some improvement in the transfer of microcolonies is necessary to increase the accuracy for gram-negative bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Combination of FT-IR microscopy and multivariate data analysis could be a complementary, rapid, and reliable tool for screening and discriminating, at species and subspecies level, micro-organisms of clinical, food-borne, or environmental origins.

Mobilization of the genetically engineered plasmid pHSV106 from Escherichia coli HB101(pHSV106) to Enterobacter cloacae in drinking water.

We have used triparental matings to demonstrate transfer (mobilization) of the nonconjugative genetically engineered plasmid pHSV106, which contains the thymidine kinase gene of herpes simplex virus cloned into pBR322, from Escherichia coli HB101 to an environmental isolate of Enterobacter cloacae in sterile drinking water. This is the first demonstration of a two-step mobilization of a genetically engineered plasmid in any type of fresh water, including drinking water. Transfer was mediated by R plasmid R100-1 of E. coli ED2149(R100-1). Matings in drinking water at 15, 25, and 35 degrees C yielded recombinants, the number of which increased with increasing temperature. Numbers of recombinants obtained were 2 orders of magnitude lower than those obtained from matings in Trypticase soy broth. High concentrations of parental organisms (2.6 x 10(8) to 2.0 x 10(9) CFU/ml) were required. During 1 week of incubation in drinking water, number of parental organisms and recombinants resulting from mobilization remained constant in the absence of indigenous organisms and declined in their presence. Using oligonucleotide probes for the cloned foreign DNA (thymidine kinase gene) and plasmid vector DNA (ampicillin resistance gene), we demonstrated that both genes were transferred to E. cloacae in the mobilization process. In one recombinant selected for detailed study, the plasmids containing these genes differed in size from all forms of pHSV106 present in E. coli HB101(pHSV106), indicating that DNA rearrangement had occurred. This recombinant maintained its plasmids in unchanged form for 15 days in drinking water. A second rearrangement occurred during serial passage of this recombinant on selective media.(ABSTRACT TRUNCATED AT 250 WORDS)

Four different genes responsible for nonimmune immunoglobulin-binding activities within a single strain of Escherichia coli.

Certain Escherichia coli strains bind the Fc fragment of immunoglobulin G (IgG) at the bacterial cell surface. Previous work established that this nonimmune Ig binding depends on several large proteins with apparent molecular masses that can exceed 200 kDa. For E. coli strain ECOR-9, four distinct genes (designated eibA, eibC, eibD, and eibE) are responsible for Ig binding. Two eib genes are linked to eaa genes, which are homologous to genes for the autotransporter family of secreted proteins. With reference to the E. coli K-12 chromosome, the eibA-eaaA cluster is adjacent to trpA (min 28.3) while the eibC-eaaC cluster is adjacent to aspS (min 42. 0). Sequence adjacent to the eibA-eaaA cluster converges with that of strain K-12 precisely as observed for the Atlas family of prophages, suggesting that eibA is part of one of these. All four eib genes, when cloned into plasmid vectors, impart IgG binding to E. coli K-12 strains, and three impart IgA binding also. The IgG binding occurs at the bacterial cell surface, and its expression increases survival in serum by up to 3 orders of magnitude. The eib sequences predict a C-terminal peptide motif that is characteristic of outer membrane proteins, and the protein sequences show significant similarity near the C terminus to both the YadA virulence factor of Yersinia species and the universal surface protein A II of Moraxella catarrhalis. The sizes predicted for Eib proteins from DNA sequence are much smaller than their apparent sizes on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, possibly reflecting stable oligomerization.

Nonimmune binding of human immunoglobulin A (IgA) and IgG Fc by distinct sequence segments of the EibF cell surface protein of Escherichia coli.

The eib genes of Escherichia coli encode surface-exposed proteins which bind immunoglobulins (Ig) such as the Fc fragment of human IgG (IgG Fc) in a nonimmune manner. The Eib proteins belong to a family which includes YadA of Yersinia, UspA2 of Moraxella, and DsrA of Haemophilus ducreyi. This family of surface-exposed proteins shares several features, such as the ability to impart resistance to human serum complement and a tendency to exist as stable multimers. Four genes, eibA, eibC, eibD and eibE, were previously identified and cloned from ECOR-9, a strain from the E. coli reference collection. EibC, -D, and -E bind human serum IgA in addition to IgG, but no IgA binding has been observed for EibA. Here, we report the cloning of a new eib gene, eibF, from a second strain of E. coli, ECOR-2. The product, EibF, has a relatively strong preference for IgA. Like the other eib genes, eibF attenuates serum sensitivity, occurs as a stable multimer, and is associated with a prophage. By subcloning portions of the eibA and eibF genes, we have identified distinct sequence segments sufficient to cause Ig binding, multimerization, and discrimination between IgA and IgG. The ability to multimerize is associated with a sequence close to the C terminus that is homologous to other family members such as YadA. Binding of IgG Fc is associated with a sequence that is highly conserved among all Eib proteins but otherwise unique. Binding of IgA is associated with a sequence of EibF that is not similar to any EibA sequence.