Ewa Bok

Structural insight into an ankyrin-sensitive lipid-binding site of erythroid β-spectrin

It was recently shown that the region within β-spectrin responsible for interactions with ankyrin includes a lipid-binding site which displayed sensitivity to inhibition by ankyrin. We studied its structure by constructing a series of single and double spin-labeled β-spectrin-derived peptides and analyzing their spin-spin distances via electron paramagnetic resonance spectroscopy and the Fourier deconvolution method. The results indicate that the whole ankyrin-sensitive lipid-binding site of β-spectrin exhibits a helical conformation revealing a distinct 310-helix contribution at its N-terminus. The start of the helix was located five residues upstream along the sequence compared to the theoretical predictions. A model based on the obtained data provides direct evidence that the examined lipid-binding site is a highly amphipathic helix, which is correlated with the specific conformation of its N-terminal fragment.

Key Amino Acid Residues of Ankyrin-Sensitive Phosphatidylethanolamine/Phosphatidylcholine-Lipid Binding Site of βI-Spectrin

It was shown previously that an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine (PE/PC) binding site maps to the N-terminal part of the ankyrin-binding domain of β-spectrin (ankBDn). Here we have identified the amino acid residues within this domain which are responsible for recognizing monolayers and bilayers composed of PE/PC mixtures. In vitro binding studies revealed that a quadruple mutant with substituted hydrophobic residues W1771, L1775, M1778 and W1779 not only failed to effectively bind PE/PC, but its residual PE/PC-binding activity was insensitive to inhibition with ankyrin. Structure prediction and analysis, supported by in vitro experiments, suggests that “opening” of the coiled-coil structure underlies the mechanism of this interaction. Experiments on red blood cells and HeLa cells supported the conclusions derived from the model and in vitro lipid-protein interaction results, and showed the potential physiological role of this binding. We postulate that direct interactions between spectrin ankBDn and PE-rich domains play an important role in stabilizing the structure of the spectrin-based membrane skeleton.

Lipid-binding role of βII-spectrin ankyrin-binding domain

It is known that erythroid and non‐erythroid spectrins binding of vesicles and monolayers containing PE proved sensitive to inhibition by red blood cell ankyrin. We now show that the bacterially‐expressed recombinant peptides representing βII(brain)‐spectrins ankyrin‐binding domain and its truncated mutants showed lipid‐binding activity, although only those containing a full‐length amino terminal fragment showed high to moderate affinity towards phospholipid mono‐ and bilayers and a substantial sensitivity of this binding to inhibition by ankyrin. These results are in accordance with our published data on βI‐spectrins ankyrin‐binding domain [Hryniewicz‐Jankowska A, et al. Mapping of ankyrin‐sensitive, PE/PC mono‐ and bilayer binding site in erythroid beta‐spectrin. Biochem J 2004;382:677–85]. Moreover, we tested also the effect of transient transfection of living cells of several cell‐lines with vectors coding for GFP‐conjugates including βII and also βI full‐length ankyrin‐binding domain and their truncated fragments on the membrane skeleton organization. The transfection with constructs encoding full‐length ankyrin‐binding domain of βII and βI spectrin resulted in increased aggregation of membrane skeleton and its punctate appearance in contrast to near normal appearance of membrane skeleton of cells transiently transfected with GFP control or construct encoding ankyrin‐binding domain truncated at their N‐terminal region. Our results therefore indicate the importance of N‐terminal region for lipid‐binding activity of the β‐spectrin ankyrin‐binding domain and its substantial role in maintaining the spectrin‐based skeleton distribution.

Antimicrobial Resistance in Commensal Escherichia coli from Pigs during Metaphylactic Trimethoprim and Sulfamethoxazole Treatment and in the Post-Exposure Period

The prevalence of trimethoprim (TMP) and sulfamethoxazole (SMX) resistance in commensal E. coli from pigs was tested in this study. E. coli was derived from three groups of piglets in successive stages of metaphylactic therapy and from two groups of sows 10 and 18 weeks after the treatment. MIC values of TMP and SMX were determined for a total of 352 strains. The presence of resistance genes (dfrA1, dfrA5, dfrA7, dfrA12, dfrA17, sul1, sul2, sul3) and class 1 and 2 integron-associated dfrA gene cassettes was tested. Resistance to TMP was very high during the administration of the antimicrobial (from 97 to 100%) and amounted to 86% and 69% in the post-exposure period; MIC > 32 mg/L. The isolates from all groups of pigs were resistant to sulfamethoxazole, with MIC > 1028 mg/L. The dfrA1 and sul1 genes (as part of integrons) dominated in E. coli from piglets, but the dfrA12 and sul1 genes were prevalent in E. coli from sows. Coexistence of the different dfrA genes was detected in 71 isolates from all groups of swine. Transcription analysis revealed that most of these genes were not transcribed, particularly gene cassettes of class 1 integrons. The research revealed a high level of resistance associated with the metaphylactic treatment, persistence and circulation of resistance in bacterial populations. Diverse genetic background with multiple and not transcribed resistance genes was observed.

Prevalence of Virulence Determinants and Antimicrobial Resistance among Commensal Escherichia coli Derived from Dairy and Beef Cattle

Cattle is a reservoir of potentially pathogenic E. coli, bacteria that can represent a significant threat to public health, hence it is crucial to monitor the prevalence of the genetic determinants of virulence and antimicrobial resistance among the E. coli population. The aim of this study was the analysis of the phylogenetic structure, distribution of virulence factors (VFs) and prevalence of antimicrobial resistance among E. coli isolated from two groups of healthy cattle: 50 cows housed in the conventional barn (147 isolates) and 42 cows living on the ecological pasture (118 isolates). The phylogenetic analysis, identification of VFs and antimicrobial resistance genes were based on either multiplex or simplex PCR. The antimicrobial susceptibilities of E. coli were examined using the broth microdilution method. Two statistical approaches were used to analyse the results obtained for two groups of cattle. The relations between the dependent (VFs profiles, antibiotics) and the independent variables were described using the two models. The mixed logit model was used to characterise the prevalence of the analysed factors in the sets of isolates. The univariate logistic regression model was used to characterise the prevalence of these factors in particular animals. Given each model, the odds ratio (OR) and the 95% confidence interval for the population were estimated. The phylogroup B1 was predominant among isolates from beef cattle, while the phylogroups A, B1 and D occurred with equal frequency among isolates from dairy cattle. The frequency of VFs-positive isolates was significantly higher among isolates from beef cattle. E. coli from dairy cattle revealed significantly higher resistance to antibiotics. Some of the tested resistance genes were present among isolates from dairy cattle. Our study showed that the habitat and diet may affect the genetic diversity of commensal E. coli in the cattle. The results suggest that the ecological pasture habitat is related to the increased spreading rate of the VFs, while the barn habitat is characterised by the higher levels of antimicrobial resistance among E. coli.

Znane i nowe warianty patogennych Escherichia coli jako konsekwencja plastycznego genomu

E. coli is a diverse bacterial species encompassing commensal as well as intestinal and extraintestinal pathogenic strains. The ability to adapt to so many different niches in the host organism is determined by the extreme genomic plasticity of E. coli. The genetic diversity is due to a complex phylogenetic structure in which besides the well-known main groups A, B1, B2 and D, four new groups, C, E, F and Clad I, have been characterized recently. The mobile gene pool exchanged by horizontal gene transfer (HGT) is another important driving force in the evolution of E. coli. Pathogenicity of strains is conditioned by a specific repertoire of virulence factors located on the mobile genetic elements and transmitted by HGT. The environment changing constantly stimulates the formation of new virulence gene combinations that generate the formation, not observed so far, of new pathogenic clones of higher capacity for virulence and greater expansiveness. The presence of very similar virulence plasmids carrying conserved combinations of the virulence genes (CVP) among extraintestinal pathogenic strains in humans and birds has been observed. It indicates a real possibility of occurrence of common virulence factors. The increase in drug resistance among pathogenic E. coli is also reflected in the prevalence of highly expansive clones exhibiting both high virulence and resistance. The presented data indicate that further studies are required to determine the interdependencies of resistance and virulence at the genetic level to help improve our management of the infectious diseases caused by these bacteria.

Chapter Four Interactions of Erythroid and Nonerythroid Spectrins and Other Membrane-Skeletal Proteins with Lipid Mono- and Bilayers

Abstract The object of the chapter is to review the studies on the interactions of erythroid and nonerythroid spectrins and other membrane-skeletal proteins with lipids in model membranes. An important progress on the identification of lipid-binding sites has recently been made although many questions remain still unanswered. In particular, our understanding of the physiological role of such interactions is still limited. Important issue is the mechanism(s) involved in these interactions.

Proteins with Spectrin Motifs Which Do Not Belong to the Spectrin-α-Actinin- Dystrophin Family

Abstract Using several consensus sequences for the 106 amino acid residue α-spectrin repeat segment as probes we searched animal sequence databases using the BLAST program in order to find proteins revealing limited, but significant similarity to spectrin. Among many spectrins and proteins from the spectrin-α-actinin-dystrophin family as well as sequences showing a rather high degree of similarity in very short stretches, we found seven homologous animal sequences of low overall similarity to spectrin but showing the presence of one or more spectrin-repeat motifs. The homology relationship of these sequences to α-spectrin was further analysed using the SEMIHOM program. Depending on the probe, these segments showed the presence of 6 to 26 identical amino acid residues and a variable number of semihomologous residues. Moreover, we found six protein sequences, which contained a sequence fragment sharing the SH3 (sarc homology region 3) domain homology of 42-59% similarity. Our data indicate the occurrence of motifs of significant homology to α-spectrin repeat segments among animal proteins, which are not classical members of the spectrin-α- actinin-dystrophin family. This might indicate that these segments together with the SH3 domain motif are conserved in proteins which possibly at the early stage of evolution were close cognates of spectrin-α-actinin-dystrophin progenitors but then evolved separately.

Comparison of Commensal Escherichia coli Isolates from Adults and Young Children in Lubuskie Province, Poland: Virulence Potential, Phylogeny and Antimicrobial Resistance

Commensal Escherichia coli population is a dynamic structure which may be important in the pathogenesis of extraintestinal infections. The aim of this study was the comparison of genetic diversity of commensal E. coli isolates from two age group—adults and young children. E. coli strains were isolated on MacConkey agar and identified by biochemical tests. Determination of four major phylogenetic groups, identification of virulence genes and antimicrobial resistance determinants were performed by using multiplex or simplex PCR. Phenotypic analysis of resistance was based on disc-diffusion method. The prevalence of virulence genes was significantly higher among isolates from adults than from young children. Phylogroup B2 predominated among E. coli from adults, whereas phylogroup A was the most common in isolates from young children. The analyses of antimicrobial resistance revealed that resistance to at least one antimicrobial agent and multidrug-resistance were detected significantly more frequent in the isolates from adults than from young children. This study documented that the commensal E. coli isolates from adults showed greater genetic diversity than from young children and constitutes a substantial reservoir of the virulence genes typical for extraintestinal pathogenic E. coli.

Phenotypic and genotypic characteristics of antibiotic resistance of commensal Escherichia coli isolates from healthy pigs

Abstract The objective of the study was to examine the characteristics of the resistance profiles of Escherichia coli isolated from healthy pigs from three farms in Western Poland. The sensitivity to 13 antimicrobial agents was tested by a disk diffusion method, and the presence of 13 resistance genes was determined by PCR. The majority of the isolates were multi-resistant. The most common multi-resistance patterns were streptomycin, trimethoprim, sulfisoxazole, ampicillin, tetracycline. Although some resistance genes, such as strA/strB, blaTEM, sul1, sul2, and tetA, were equally represented in isolates from each farm, differences in the distribution of tetB and tetC, hfrV, dhfrXII, and sul1 resistance genes were observed among the isolates from different farms. Approximately one-third (35.9%) of the isolates possessed a class 1 integron. The four major different variable regions of the class 1 integron contained streptomycin (aadA1, aadA2, and aadA5) and/or trimethoprim (dhfrI, dhfrV and dhfrXVII), and/or sulphonamides (sul1) resistance genes. The results of this study emphasise that uncontrolled use of antibiotics causes the development of resistance and provides the evidence of frequent occurrence of more than one gene encoding the resistance to the same antimicrobial agent in the multi-resistant strains.