Studies of antibacterial activity (in vitro and in vivo) and mode of action for des-acyl tridecaptins (DATs).

Tridecaptins comprise a class of linear cationic lipopeptides with an N-terminal fatty acyl moiety. These 13-mer antimicrobial peptides consist of a combination of d- and l-amino acids, conferring increased proteolytic stability. Intriguingly, they are biosynthesized by non-ribosomal peptide synthetases in the same bacterial species that also produce the cyclic polymyxins displaying similar fatty acid tails. Previously, the des-acyl analog of TriA1 (termed H-TriA1) was found to possess very weak antibacterial activity, albeit it potentiated the effect of several antibiotics. In the present study, two series of des-acyl tridecaptins were explored with the aim of improving the direct antibacterial effect. At the same time, overall physico-chemical properties were modulated by amino acid substitution(s) to diminish the risk of undesired levels of hemolysis and to avoid an impairment of mammalian cell viability, since these properties are typically associated with highly hydrophobic cationic peptides. Microbiology and biophysics tools were used to determine bacterial uptake, while circular dichroism and isothermal calorimetry were used to probe the mode of action. Several analogs had improved antibacterial activity (as compared to that of H-TriA1) against Enterobacteriaceae. Optimization enabled identification of the lead compound 29 that showed a good ADMET profile as well as in vivo efficacy in a variety of mouse models of infection.

Full Profiling of GE81112A, an Underexplored Tetrapeptide Antibiotic with Activity against Gram-Negative Pathogens.

After the first total synthesis combined with structure revision, we performed thorough in vitro and in vivo profiling of the underexplored tetrapeptide GE81112A. From the determination of the biological activity spectrum and physicochemical and early absorption-distribution-metabolism-excretion-toxicity (eADMET) properties, as well as in vivo data regarding tolerability and pharmacokinetics (PK) in mice and efficacy in an Escherichia coli-induced septicemia model, we were able to identify the critical and limiting parameters of the original hit compound. Thus, the generated data will serve as the basis for further compound optimization programs and developability assessments to identify candidates for preclinical/clinical development derived from GE81112A as the lead structure. IMPORTANCE The spread of antimicrobial resistance (AMR) is becoming a more and more important global threat to human health. With regard to current medical needs, penetration into the site of infection represents the major challenge in the treatment of infections caused by Gram-positive bacteria. Considering infections associated with Gram-negative bacteria, resistance is a major issue. Obviously, novel scaffolds for the design of new antibacterials in this arena are urgently needed to overcome this crisis. Such a novel potential lead structure is represented by the GE81112 compounds, which inhibit protein synthesis by interacting with the small 30S ribosomal subunit using a binding site distinct from that of other known ribosome-targeting antibiotics. Therefore, the tetrapeptide antibiotic GE81112A was chosen for further exploration as a potential lead for the development of antibiotics with a new mode of action against Gram-negative bacteria.

Structure Elucidation, Total Synthesis, Antibacterial In Vivo Efficacy and Biosynthesis Proposal of Myxobacterial Corramycin.

Herein, we describe the myxobacterial natural product Corramycin isolated from Corallococcus coralloides. The linear peptide structure contains an unprecedented (2R,3S)-γ-N-methyl-ß-hydroxy-histidine moiety. Corramycin exhibits anti-Gram-negative activity against Escherichia coli (E. coli) and is taken up via two transporter systems, SbmA and YejABEF. Furthermore, the Corramycin biosynthetic gene cluster (BGC) was identified and a biosynthesis model was proposed involving a 12-modular non-ribosomal peptide synthetase/polyketide synthase. Bioinformatic analysis of the BGC combined with the development of a total synthesis route allowed for the elucidation of the molecule's absolute configuration. Importantly, intravenous administration of 20 mg kg-1 of Corramycin in an E. coli mouse infection model resulted in 100 % survival of animals without toxic side effects. Corramycin is thus a promising starting point to develop a potent antibacterial drug against hospital-acquired infections.

Antibiotics and efflux: combined spectrofluorimetry and mass spectrometry to evaluate the involvement of concentration and efflux activity in antibiotic intracellular accumulation.

Background: In Gram-negative bacteria, passing through the double membrane barrier to reach the inhibitory concentration inside the bacterium is a pivotal step for antibiotic activity. Spectrofluorimetry has been developed to follow fluoroquinolone accumulation inside bacteria using intrinsic bacterial fluorescence as an internal standard. However, adaptation for non-fluorescent antibiotics is needed; quantitative methods based on MS offer the possibility of expanding the detection range obtained by spectrofluorimetry. Objectives: To validate, with spectrofluorimetry, the use of MS to measure antibiotic accumulation in cells and to determine the relationship between antibiotic concentrations and the amount of intrabacterial accumulation in different efflux backgrounds on the same batch of molecules. Methods: Spectrofluorimetry was performed in parallel with MS on the same samples to measure the ciprofloxacin and fleroxacin accumulation in cells expressing various efflux pump levels. A microplate protocol was set up to determine the antibiotic accumulation as a function of external antibiotic concentrations. Results: A correlation existed between the data obtained with spectrofluorimetry and MS, whatever the efflux pump or tested antibiotic. The results highlighted different dynamics of uptake between ciprofloxacin and fleroxacin as well as the relationship between the level of efflux activity and antibiotic accumulation. Conclusions: We have developed a microplate protocol and cross-validated two complementary methods: spectrofluorimetry, which contains a reliable internal standard; and MS, which allows detection of low antibiotic amounts. These assays allow study of the dose effect and the efflux impact on the intrabacterial accumulation of antibiotics.

Expression of the human antimicrobial peptide ß-defensin-1 is repressed by the EGFR-ERK-MYC axis in colonic epithelial cells.

The human ß-defensin-1 (HBD1) is an antimicrobial peptide constitutively expressed by epithelial cells at mucosal surfaces. In addition to its microbicidal properties, the loss of HBD1 expression in several cancers suggests that it may also have an anti-tumor activity. Here, we investigated the link between HBD1 expression and cancer signaling pathways in the human colon cancer cell lines TC7 and HT-29, and in normal human colonic primary cells, using a mini-gut organoid model. Using available datasets from patient cohorts, we found that HBD1 transcription is decreased in colorectal cancer. We demonstrated that inhibiting the Epidermal Growth Factor Receptor (EGFR) increased HBD1 expression, whereas activating EGFR repressed HBD1 expression, through the MEKK1/2-ERK1/2 pathway that ultimately regulates MYC. We finally present evidences supporting a role of MYC, together with the MIZ1 coregulator, in HBD1 regulation. Our work uncovers the role and deciphers the function of the EGFR-ERK-MYC axis as a repressor of HBD1 expression and contributes to the understanding of HBD1 suppression observed in colorectal cancer.

Imidazopyrazinones (IPYs): Non-Quinolone Bacterial Topoisomerase Inhibitors Showing Partial Cross-Resistance with Quinolones.

In our quest for new antibiotics able to address the growing threat of multidrug resistant infections caused by Gram-negative bacteria, we have investigated an unprecedented series of non-quinolone bacterial topoisomerase inhibitors from the Sanofi patrimony, named IPYs for imidazopyrazinones, as part of the Innovative Medicines Initiative (IMI) European Gram Negative Antibacterial Engine (ENABLE) organization. Hybridization of these historical compounds with the quinazolinediones, a known series of topoisomerase inhibitors, led us to a novel series of tricyclic IPYs that demonstrated potential for broad spectrum activity, in vivo efficacy, and a good developability profile, although later profiling revealed a genotoxicity risk. Resistance studies revealed partial cross-resistance with fluoroquinolones (FQs) suggesting that IPYs bind to the same region of bacterial topoisomerases as FQs and interact with at least some of the keys residues involved in FQ binding.

Fluoroquinolone structure and translocation flux across bacterial membrane.

Bacterial multidrug resistance is a worrying health issue. In Gram-negative antibacterial research, the challenge is to define the antibiotic permeation across the membranes. Passing through the membrane barrier to reach the inhibitory concentration inside the bacterium is a pivotal step for antibacterial molecules. A spectrofluorimetric methodology has been developed to detect fluoroquinolones in bacterial population and inside individual Gram-negative bacterial cells. In this work, we studied the antibiotic accumulation in cells expressing various levels of efflux pumps. The assays allow us to determine the intracellular concentration of the fluoroquinolones to study the relationships between the level of efflux activity and the antibiotic accumulation, and finally to evaluate the impact of fluoroquinolone structures in this process. This represents the first protocol to identify some structural parameters involved in antibiotic translocation and accumulation, and to illustrate the recently proposed "Structure Intracellular Concentration Activity Relationship" (SICAR) concept.

Chromosomal position shift of a regulatory gene alters the bacterial phenotype.

Recent studies strongly suggest that in bacterial cells the order of genes along the chromosomal origin-to-terminus axis is determinative for regulation of the growth phase-dependent gene expression. The prediction from this observation is that positional displacement of pleiotropic genes will affect the genetic regulation and hence, the cellular phenotype. To test this prediction we inserted the origin-proximal dusB-fis operon encoding the global regulator FIS in the vicinity of replication terminus on both arms of the Escherichia coli chromosome. We found that the lower fis gene dosage in the strains with terminus-proximal dusB-fis operons was compensated by increased fis expression such that the intracellular concentration of FIS was homeostatically adjusted. Nevertheless, despite unchanged FIS levels the positional displacement of dusB-fis impaired the competitive growth fitness of cells and altered the state of the overarching network regulating DNA topology, as well as the cellular response to environmental stress, hazardous substances and antibiotics. Our finding that the chromosomal repositioning of a regulatory gene can determine the cellular phenotype unveils an important yet unexplored facet of the genetic control mechanisms and paves the way for novel approaches to manipulate bacterial physiology.

The Pseudomonas aeruginosa PA14 ABC Transporter NppA1A2BCD Is Required for Uptake of Peptidyl Nucleoside Antibiotics.

UNLABELLED: Analysis of the genome sequence of Pseudomonas aeruginosa PA14 revealed the presence of an operon encoding an ABC-type transporter (NppA1A2BCD) showing homology to the Yej transporter of Escherichia coli. The Yej transporter is involved in the uptake of the peptide-nucleotide antibiotic microcin C, a translation inhibitor that targets the enzyme aspartyl-tRNA synthetase. Furthermore, it was recently shown that the Opp transporter from P. aeruginosa PAO1, which is identical to Npp, is required for uptake of the uridyl peptide antibiotic pacidamycin, which targets the enzyme translocase I (MraY), which is involved in peptidoglycan synthesis. We used several approaches to further explore the substrate specificity of the Npp transporter. Assays of growth in defined minimal medium containing peptides of various lengths and amino acid compositions as sole nitrogen sources, as well as Biolog Phenotype MicroArrays, showed that the Npp transporter is not required for di-, tri-, and oligopeptide uptake. Overexpression of the npp operon increased susceptibility not just to pacidamycin but also to nickel chloride and the peptidyl nucleoside antibiotic blasticidin S. Furthermore, heterologous expression of the npp operon in a yej-deficient mutant of E. coli resulted in increased susceptibility to albomycin, a naturally occurring sideromycin with a peptidyl nucleoside antibiotic. Additionally, heterologous expression showed that microcin C is recognized by the P. aeruginosa Npp system. Overall, these results suggest that the NppA1A2BCD transporter is involved in the uptake of peptidyl nucleoside antibiotics by P. aeruginosa PA14. IMPORTANCE: One of the world's most serious health problems is the rise of antibiotic-resistant bacteria. There is a desperate need to find novel antibiotic therapeutics that either act on new biological targets or are able to bypass known resistance mechanisms. Bacterial ABC transporters play an important role in nutrient uptake from the environment. These uptake systems could also be exploited by a Trojan horse strategy to facilitate the transport of antibiotics into bacterial cells. Several natural antibiotics mimic substrates of peptide uptake routes. In this study, we analyzed an ABC transporter involved in the uptake of nucleoside peptidyl antibiotics. Our data might help to design drug conjugates that may hijack this uptake system to gain access to cells.

High-throughput screening of dipeptide utilization mediated by the ABC transporter DppBCDF and its substrate-binding proteins DppA1-A5 in Pseudomonas aeruginosa.

In this study, we show that the dppBCDF operon of Pseudomonas aeruginosa PA14 encodes an ABC transporter responsible for the utilization of di/tripeptides. The substrate specificity of ABC transporters is determined by its associated substrate-binding proteins (SBPs). Whereas in E. coli only one protein, DppA, determines the specificity of the transporter, five orthologous SBPs, DppA1-A5 are present in P. aeruginosa. Multiple SBPs might broaden the substrate specificity by increasing the transporter capacity. We utilized the Biolog phenotype MicroArray technology to investigate utilization of di/tripeptides in mutants lacking either the transport machinery or all of the five SBPs. This high-throughput method enabled us to screen hundreds of dipeptides with various side-chains, and subsequently, to determine the substrate profile of the dipeptide permease. The substrate spectrum of the SBPs was elucidated by complementation of a penta mutant, deficient of all five SBPs, with plasmids carrying individual SBPs. It became apparent that some dipeptides were utilized with different affinity for each SBP. We found that DppA2 shows the highest flexibility on substrate recognition and that DppA2 and DppA4 have a higher tendency to utilize tripeptides. DppA5 was not able to complement the penta mutant under our screening conditions. Phaseolotoxin, a toxic tripeptide inhibiting the enzyme ornithine carbamoyltransferase, is also transported into P. aeruginosa via the DppBCDF permease. The SBP DppA1, and with much greater extend DppA3, are responsible for delivering the toxin to the permease. Our results provide a first overview of the substrate pattern of the ABC dipeptide transport machinery in P. aeruginosa.

Leucine-responsive regulatory protein Lrp and PapI homologues influence phase variation of CS31A fimbriae.

CS31A, a K88-related surface antigen specified by the clp operon, is a member of the type P family of adhesive factors and plays a key role in the establishment of disease caused by septicemic and enterotoxigenic Escherichia coli strains. Its expression is under the control of methylation-dependent transcriptional regulation, for which the leucine-responsive regulatory protein (Lrp) is essential. CS31A is preferentially in the OFF state and exhibits distinct regulatory features compared to the regulation of other P family members. In the present study, surface plasmon resonance and DNase I protection assays showed that Lrp binds to the distal moiety of the clp regulatory region with low micromolar affinity compared to its binding to the proximal moiety, which exhibits stronger, nanomolar affinity. The complex formation was also influenced by the addition of PapI or FooI, which increased the affinity of Lrp for the clp distal and proximal regions and was required to induce phase variation. The influence of PapI or FooI, however, was predominantly associated with a more complete shutdown of clp expression, in contrast to what has previously been observed with AfaF (a PapI ortholog). Taken together, these results suggest that the preferential OFF state observed in CS31A cells is mainly due to the weak interaction of the leucine-responsive regulatory protein with the clp distal region and that the PapI homolog favors the OFF phase. Within the large repertoire of fimbrial variants in the P family, our study illustrates that having a fimbrial operon that lacks its own PapI ortholog allows it to be more flexibly regulated by other orthologs in the cell.

Glycosylation of the Escherichia coli TibA self-associating autotransporter influences the conformation and the functionality of the protein.

The self-associating autotransporters (SAATs) are multifunctional secreted proteins of Escherichia coli, comprising the AIDA-I, TibA and Ag43 proteins. One of their characteristics is that they can be glycosylated. Glycosylation of AIDA-I and Ag43 have been investigated, but not that of TibA. It is still not clear whether glycosylation of the SAATs affect their structure or their functionality. Therefore, we have looked at the effects of glycosylation on the TibA adhesin/invasin. TibA is glycosylated by TibC, a specific glycosyltransferase, and the two genes are encoded in an operon. In this study, we have found that the glycosylation of TibA is not limited to the extracellular functional domain, as previously observed with AIDA-I and Ag43. We have determined that unglycosylated TibA is not able to promote the adhesion of bacteria on cultured epithelial cell, even though it is still able to promote invasion, biofilm formation and autoaggregation of bacteria. We have purified the glycosylated and unglycosylated forms of TibA, and determined that TibA is less stable when not glycosylated. We finally observed that glycosylation affects the oligomerisation of TibA and that unglycosylated TibA is locked in a conformation that is not suited for adhesion. Our results suggest that the effect of glycosylation on the functionality of TibA is indirect.

Effects of sterilization with hydrogen peroxide gas plasma, ethylene oxide, and steam on bioadhesive properties of nylon and polyethylene lines used for stabilization of canine stifle joints.

OBJECTIVE: To compare effects of sterilization with hydrogen peroxide gas plasma (HPGP), ethylene oxide, and steam on bioadhesive properties of nylon and polyethylene lines used for stabilization of canine stifle joints. SAMPLE: Samples of a 36.3-kg test nylon leader line, 57.8-kg test nylon fishing line, and 2-mm ultrahigh-molecular weight polyethylene (UHMWPE) were used. PROCEDURES: In this in vitro study, samples of nylon leader line, fishing line, and UHMWPE sterilized by use of HPGP, ethylene oxide, and steam or unsterilized samples were used. Bacterial adherence on unsterilized and sterilized samples was tested with Staphylococcus epidermidis and Escherichia coli. Five samples were examined for each line type and sterilization condition, and final colony counts were obtained. RESULTS: Bacterial adherence was significantly affected by method of sterilization for all 3 line types. For most of the samples, bacterial adherence was similar or lower when HPGP sterilization was used, compared with results for sterilization via ethylene oxide and steam, respectively. Bacterial adherence was significantly higher for UHMWPE, compared with adherence for the nylon line, regardless of the sterilization method used. Bacterial adherence was higher for nylon fishing line than for nylon leader line for S epidermidis after ethylene oxide sterilization and for E coli after HPGP and ethylene oxide sterilization. CONCLUSIONS AND CLINICAL RELEVANCE: Effects of HPGP sterilization on bioadhesive properties of nylon and polyethylene lines compared favorably with those for ethylene oxide and steam sterilization. Also, nylon line may be a more suitable material than UHMWPE for suture prostheses on the basis of bacterial adherence properties.

Decreased expression of type 1 fimbriae by a pst mutant of uropathogenic Escherichia coli reduces urinary tract infection.

The pstSCAB-phoU operon encodes the phosphate-specific transport system (Pst). Loss of Pst constitutively activates the Pho regulon and decreases bacterial virulence. However, specific mechanisms underlying decreased bacterial virulence through inactivation of Pst are poorly understood. In uropathogenic Escherichia coli (UPEC) strain CFT073, inactivation of pst decreased urinary tract colonization in CBA/J mice. The pst mutant was deficient in production of type 1 fimbriae and showed decreased expression of the fimA structural gene which correlated with differential expression of the fimB, fimE, ipuA, and ipbA genes, encoding recombinases, mediating inversion of the fim promoter. The role of fim downregulation in attenuation of the pst mutant was confirmed using a fim phase-locked-on derivative, which demonstrated a significant gain in virulence. In addition, the pst mutant was less able to invade human bladder epithelial cells. Since type 1 fimbriae contribute to UPEC virulence by promoting colonization and invasion of bladder cells, the reduced bladder colonization by the pst mutant is predominantly attributed to downregulation of these fimbriae. Elucidation of mechanisms mediating the control of type 1 fimbriae through activation of the Pho regulon in UPEC may open new avenues for therapeutics or prophylactics against urinary tract infections.

Identification and mechanism of evolution of new alleles coding for the AIDA-I autotransporter of porcine pathogenic Escherichia coli.

Autotransporters are a large family of virulence factors of Gram-negative bacterial pathogens. The autotransporter adhesin involved in diffuse adherence (AIDA-I) is an outer membrane protein of Escherichia coli, which allows binding to epithelial cells as well as the autoaggregation of bacteria. AIDA-I is glycosylated by a specific heptosyltransferase encoded by the aah gene that forms an operon with the aidA gene. aidA is highly prevalent in strains that cause disease in pigs. Nevertheless, there are only two published whole-length sequences for this gene. In this study, we sequenced the aah and aidA genes of 24 aidA-positive porcine strains harboring distinct virulence factor profiles. We compared the obtained sequences and performed phylogenetic and pulsed-field electrophoresis analyses. Our results suggest that there are at least 3 different alleles for aidA, which are associated with distinct virulence factor profiles. The genes are found on high-molecular-weight plasmids and seem to evolve via shuffling mechanisms, with one of the sequences showing evidence of genetic recombination. Our work suggests that genetic plasticity allows the evolution of aah-aidA alleles that are selected during pathogenesis.

A structural motif is the recognition site for a new family of bacterial protein O-glycosyltransferases.

The Escherichia coli Adhesin Involved in Diffuse Adherence (AIDA-I) is a multifunctional protein that belongs to the family of monomeric autotransporters. This adhesin can be glycosylated by the AIDA-associated heptosyltransferase (Aah). Glycosylation appears to be restricted to the extracellular domain of AIDA-I, which comprises imperfect repeats of a 19-amino-acid consensus sequence and is predicted to form a ß-helix. Here, we show that Aah homologues can be found in many Gram-negative bacteria, including Citrobacter rodentium. We demonstrated that an AIDA-like protein is glycosylated in this species by the Aah homologue. We then investigated the substrate recognition mechanism of the E. coli Aah heptosyltransferase. We found that a peptide corresponding to one repeat of the 19-amino-acid consensus is sufficient for recognition and glycosylation by Aah. Mutagenesis studies suggested that, unexpectedly, Aah recognizes a structural motif typical of ß-helices, but not a specific sequence. In agreement with this finding, we observed that the extracellular domain of the Bordetella pertussis pertactin, a ß-helical polypeptide lacking the 19-amino-acid consensus sequence, could be glycosylated by Aah. Overall, our findings suggest that Aah represents the prototype of a new large family of bacterial protein O-glycosyltransferases that modify various substrates recognized through a structural motif.

Biochemical and biological characterization of Escherichia coli STb His12 to Asn variant.

We identified a variant of Escherichia coli STb toxin by PCR amplification of clinical isolates obtained from diseased pigs. The variant differed by only one amino acid at position 12 from His to Asn. This change was observed in 23 of the 100 randomly selected enterotoxigenic E. coli (ETEC) isolates tested. There was a positive correlation between the presence of the STa enterotoxin and the STb variant. As the variant represented a high percentage of the ETEC strains tested, we were interested in determining if the single amino acid change results in altered biological characteristics of the toxin. Circular dichroism analysis revealed that the secondary structure of the variant was similar to wildtype and that their thermal stabilities were similar. Surface plasmon resonance showed that the variant and the wildtype toxins possessed similar binding affinities for sulfatide but the variant exhibited a reduced binding capacity. A flow cytometry-based internalization assay showed that the variant toxin is more internalized into epithelial intestinal cells than the wildtype strain. However, this difference was minor. Overall, our results indicate that while wildtype STb and the variant share similar structural properties, modest differences exist in their internalization.

Lrp-DNA complex stability determines the level of ON cells in type P fimbriae phase variation.

F165(1) and the pyelonephritis-associated pili (Pap) are two members of the type P family of adhesive factors that play a key role in the establishment of disease caused by extraintestinal Escherichia coli (ExPEC) strains. They are both under the control of an epigenetic and reversible switch that defines the number of fimbriated (ON) and afimbriated (OFF) cells within a clonal population. Our present study demonstrates that the high level of ON cells found during F165(1) phase variation is due to altered stability of the DNA complex formed by the leucine-responsive regulatory protein (Lrp) at its repressor binding sites 1-3; after each cell cycle, complex formation is also modulated by the local regulator FooI (homologue to PapI) which promotes the transit of Lrp towards its activator binding sites 4-6. Furthermore, we identified two nucleotides (T490, G508) surrounding the Lrp binding site 1 that are critical to maintaining a high OFF to ON switch rate during F165(1) phase variation, as well as switching Pap fimbriae towards the OFF state.

In vitro assay of bacterial adhesion onto mammalian epithelial cells.

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells(1). These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'(2). Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories(3,4). These assays are now practiced by most laboratories working on bacterial pathogenesis. Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 2787(5), a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787ΔaidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before(6).

Structure-function analysis of the TibA self-associating autotransporter reveals a modular organization.

Some enterotoxigenic Escherichia coli strains express the TibA adhesin/invasin, a multifunctional autotransporter that mediates the autoaggregation of bacteria, biofilm formation, adhesion to cultured epithelial cells, and invasion of these cells. To elucidate the structure-function relationship in TibA, we generated mutants by transposon-based linker scanning mutagenesis and by site-directed mutagenesis. Several insertion mutants had a defect in either adhesion or autoaggregation. Mutants with a defect in autoaggregation were found in the N-terminal half of the extracellular domain, while mutants with a defect in adhesion were found in the C-terminal half. The deletion of the putative N-terminal autoaggregation domain abolished the autoaggregation of the bacteria but did not affect adhesion. The deletion of a proline-rich region located at the C terminus of the extracellular domain abolished the adhesion properties of TibA but did not affect invasion. This finding suggests that adhesion and invasion may rely on distinct mechanisms. Thus, our results reveal that TibA possesses a modular organization, with the extracellular domain being separated into an autoaggregation module and an adhesion module.