Role of the YehD fimbriae in the virulence-associated properties of enteroaggregative Escherichia coli.

The interaction of enteroaggregative Escherichia coli (EAEC) strains with the colonic gut mucosa is characterized by the ability of the bacteria to form robust biofilms, to bind mucin, and induce a local inflammatory response. These events are mediated by a repertoire of five different aggregative adherence fimbriae variants (AAF/I-V) typically encoded on virulence plasmids. In this study, we report the production in EAEC strains of a new YehD fimbriae (YDF), which is encoded by the chromosomal gene cluster yehABCD, also present in most E. coli strains. Immuno-labelling of EAEC strain 042 with anti-AAF/II and anti-YDF antibodies demonstrated the presence of both AAF/II and YDF on the bacterial surface. We investigated the role of YDF in cell adherence, biofilm formation, colonization of spinach leaves, and induction of pro-inflammatory cytokines release. To this aim, we constructed yehD deletion mutants in different EAEC backgrounds (strains 17-2, 042, 55989, C1010, 278-1, J7) each harbouring one of the five AAFs. The effect of the YDF mutation was strain dependent and AAF independent as the lack of YDF had a different impact on the phenotypes manifested by the different EAECs tested. Expression of the yehABCD operon in a E. coli K12 ORN172 showed that YDF is important for biofilm formation but not for adherence to HeLa cells. Lastly, screening of pro-inflammatory cytokines in supernatants of Caco-2 cells infected with EAEC strains 042 and J7 and their isogenic ΔyehD mutants showed that these mutants were significantly defective in release of IL-8 and TNF-α. This study contributes to the understanding of the complex and diverse mechanisms of adherence of EAEC strains and identifies a new potential target for preventive measures of gastrointestinal illness caused by EAEC and other E. coli pathogroups.

The Assembly of Flagella in Enteropathogenic Escherichia coli Requires the Presence of a Functional Type III Secretion System.

In enteropathogenic Escherichia coli (EPEC), the production of flagella and the type III secretion system (T3SS) is activated in the presence of host cultured epithelial cells. The goal of this study was to investigate the relationship between expression of flagella and the T3SS. Mutants deficient in assembling T3SS basal and translocon components (ΔespA, ΔespB, ΔespD, ΔescC, ΔescN, and ΔescV), and in secreting effector molecules (ΔsepD and ΔsepL) were tested for flagella production under several growth conditions. The ΔespA mutant did not produce flagella in any condition tested, although fliC was transcribed. The remaining mutants produced different levels of flagella upon growth in LB or in the presence of cells but were significantly diminished in flagella production after growth in Dulbecco's minimal essential medium. We also investigated the role of virulence and global regulator genes in expression of flagella. The ΔqseB and ΔqseC mutants produced abundant flagella only when growing in LB and in the presence of HeLa cells, indicating that QseB and QseC act as negative regulators of fliC transcription. The ΔgrlR, ΔperA, Δler, Δhns, and Δfis mutants produced low levels of flagella, suggesting these regulators are activators of fliC expression. These data suggest that the presence of an intact T3SS is required for assembly of flagella highlighting the existence in EPEC of a cross-talk between these two virulence-associated T3SSs.

The EcpD Tip Adhesin of the Escherichia coli Common Pilus Mediates Binding of Enteropathogenic E. coli to Extracellular Matrix Proteins.

The attachment of enteropathogenic Escherichia coli (EPEC) to intestinal epithelial cells is facilitated by several adhesins; however, the individual host-cell receptors for pili-mediated adherence have not been fully characterized. In this study, we evaluated the hypothesis that the E. coli common pilus (ECP) tip adhesin protein EcpD mediates attachment of EPEC to several extracellular matrix (ECM) glycoproteins (fibronectin, laminin, collagens I and IV, and mucin). We found that the ΔecpA mutant, which lacks production of the EcpA filament but retains EcpD on the surface, adhered to these glycoproteins below the wild-type levels, while the ΔecpD mutant, which does not display EcpA or EcpD, bound significantly less to these host glycoproteins. In agreement, a purified recombinant EcpD subunit bound significantly more than EcpA to laminin, fibronectin, collagens I and IV, and mucin in a dose-dependent manner. These are compelling data that strongly suggest that ECP-producing EPEC may bind to host ECM glycoproteins and mucins through the tip adhesin protein EcpD. This study highlights the versatility of EPEC to bind to different host proteins and suggests that the interaction of ECP with the host's ECM glycoproteins may facilitate colonization of the intestinal mucosal epithelium.

Aggregative Adherence Fimbriae II of Enteroaggregative Escherichia coli Are Required for Adherence and Barrier Disruption during Infection of Human Colonoids.

Symptomatic and asymptomatic infection with the diarrheal pathogen enteroaggregative Escherichia coli (EAEC) is associated with growth faltering in children in developing settings. The mechanism of this association is unknown, emphasizing a need for better understanding of the interactions between EAEC and the human gastrointestinal mucosa. In this study, we investigated the role of the aggregative adherence fimbriae II (AAF/II) in EAEC adherence and pathogenesis using human colonoids and duodenal enteroids. We found that a null mutant in aafA, the major subunit of AAF/II, adhered significantly less than wild-type (WT) EAEC strain 042, and adherence was restored in a complemented strain. Immunofluorescence confocal microscopy of differentiated colonoids, which produce an intact mucus layer comprised of the secreted mucin MUC2, revealed bacteria at the epithelial surface and within the MUC2 layer. The WT strain adhered to the epithelial surface, whereas the aafA deletion strain remained within the MUC2 layer, suggesting that the presence or absence of AAF/II determines both the abundance and location of EAEC adherence. In order to determine the consequences of EAEC adherence on epithelial barrier integrity, colonoid monolayers were exposed to EAEC constructs expressing or lacking aafA Colonoids infected with WT EAEC had significantly decreased epithelial resistance, an effect that required AAF/II, suggesting that binding of EAEC to the epithelium is necessary to impair barrier function. In summary, we show that production of AAF/II is critical for adherence and barrier disruption in human colonoids, suggesting a role for this virulence factor in EAEC colonization of the gastrointestinal mucosa.

The CpxRA stress response system regulates virulence features of avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) causes localized and systemic avian infections and is responsible for considerable economic losses in the poultry industry. This organism adheres and invades human and avian cells, however, the regulatory networks that dictate its virulence are largely unknown. The CpxRA two-component system is responsible for sensing and controlling outer-membrane stress and detecting misfolded proteins in the bacterial periplasmic space. CpxA is a membrane sensor kinase and CpxR is a cytoplasmic transcriptional regulator. In this study, we found that the CpxRA system regulates the virulence properties of APEC. Adherence, invasiveness, motility, production of type 1 fimbriae and biofilm were negatively affected in the ΔcpxA mutant indicating that the CpxA is required for full manifestation of these phenotypes. We also found that CpxR-P directly bound to the fimA promoter, locking the fimS region of type 1 fimbriae in the phase-OFF orientation. In addition, the absence of CpxA also reduced flagella production strongly suggesting that CpxRA regulates these two important surface organelles in APEC. This study provides compelling evidence of the role of the CpxRA two-component system in the regulation of virulence factors of avian pathogenic E. coli.

The expression of Longus type 4 pilus of enterotoxigenic Escherichia coli is regulated by LngR and LngS and by H-NS, CpxR and CRP global regulators.

Enterotoxigenic Escherichia coli produces a long type 4 pilus called Longus. The regulatory elements and the environmental signals controlling the expression of Longus-encoding genes are unknown. We identified two genes lngR and lngS in the Longus operon, whose predicted products share homology with transcriptional regulators. Isogenic lngR and lngS mutants were considerably affected in transcription of lngA pilin gene. The expression of lngA, lngR and lngS genes was optimally expressed at 37°C at pH 7.5. The presence of glucose and sodium chloride had a positive effect on Longus expression. The presence of divalent ions, particularly calcium, appears to be an important stimulus for Longus production. In addition, we studied H-NS, CpxR and CRP global regulators, on Longus expression. The response regulator CpxR appears to function as a positive regulator of lng genes as the cpxR mutant showed reduced levels of lngRSA expression. In contrast, H-NS and CRP function as negative regulators since expression of lngA was up-regulated in isogenic hns and crp mutants. H-NS and CRP were required for salt- and glucose-mediated regulation of Longus. Our data suggest the existence of a complex regulatory network controlling Longus expression, involving both local and global regulators in response to different environmental signals.

Curli Temper Adherence of Escherichia coli O157:H7 to Squamous Epithelial Cells from the Bovine Recto-Anal Junction in a Strain-Dependent Manner.

Our recent studies have shown that intimin and the locus of enterocyte effacement-encoded proteins do not play a role in Escherichia coli O157:H7 (O157) adherence to the bovine recto-anal junction squamous epithelial (RSE) cells. To define factors that play a contributory role, we investigated the role of curli, fimbrial adhesins commonly implicated in adherence to various fomites and plant and human epithelial cells, in O157 adherence to RSE cells. Specifically, we examined (i) wild-type strains of O157; (ii) curli variants of O157 strains; (iii) isogenic curli deletion mutants of O157; and (iv) adherence inhibition of O157 using anti-curlin sera. Results of these experiments conducted under stringent conditions suggest that curli do not solely contribute to O157 adherence to RSE cells and in fact demonstrate a modulating effect on O157 adherence to RSE cells in contrast to HEp-2 cells (human epidermoid carcinoma of the larynx cells with HeLa contamination). The absence of curli and presence of blocking anti-curli antibodies enhanced O157-RSE cell interactions among some strains, thus alluding to a spatial, tempering effect of curli on O157 adherence to RSE cells when present. At the same time, the presence or absence of curli did not alter RSE cell adherence patterns of another O157 strain. These observations are at variance with the reported role of curli in O157 adherence to human cell lines such as HEp-2 and need to be factored in when developing anti-adherence modalities for preharvest control of O157 in cattle. IMPORTANCE: This study demonstrated that O157 strains interact with epithelial cells in a host-specific manner. The fimbriae/adhesins that are significant for adherence to human cell lines may not have a role or may have a modulating role in O157 adherence to bovine cells. Targeting such adhesins may not prevent O157 attachment to bovine cells but instead may result in improved adherence. Hence, conducting host-specific evaluations is critical when selecting targets for O157 control strategies.

Structural insights into the biogenesis and biofilm formation by the Escherichia coli common pilus.

Bacteria have evolved a variety of mechanisms for developing community-based biofilms. These bacterial aggregates are of clinical importance, as they are a major source of recurrent disease. Bacterial surface fibers (pili) permit adherence to biotic and abiotic substrates, often in a highly specific manner. The Escherichia coli common pilus (ECP) represents a remarkable family of extracellular fibers that are associated with both disease-causing and commensal strains. ECP plays a dual role in early-stage biofilm development and host cell recognition. Despite being the most common fimbrial structure, relatively little is known regarding its biogenesis, architecture, and function. Here we report atomic-resolution insight into the biogenesis and architecture of ECP. We also derive a structural model for entwined ECP fibers that not only illuminates interbacteria communication during biofilm formation but also provides a useful foundation for the design of novel nanofibers.

OmpR phosphorylation regulates ompS1 expression by differentially controlling the use of promoters.

The Salmonella enterica ompS1 gene encodes a quiescent porin that belongs to the OmpC/OmpF family. In the present work we analysed the regulatory effects of OmpR phosphorylation on ompS1 expression. We found that in vivo, OmpR in its phosphorylated form (OmpR-P) was important in the regulation of the two ompS1 promoters: OmpR-P activated the P1 promoter and repressed the P2 promoter in an EnvZ-dependent manner; expression occurs from the P2 promoter in an ompR mutant. In vitro, OmpR-P had a higher DNA-binding-affinity to the ompS1 promoter region than OmpR and OmpRD55A, showing an affinity even higher than that of equivalent DNA regions in the 5'-upstream regulatory sequence of the major porin-encoding genes ompC and ompF. By analysing different environmental conditions, we found that glucose and glycerol enhanced ompS1 expression in the wild-type strain. Interestingly the stimulation by glycerol was OmpR-dependent while the effect of glucose was still observed in the absence of OmpR. Acetyl phosphate produced by the AckA-Pta pathway did not influence ompS1 regulation. These data indicate the important role of the phosphorylation in the activity of OmpR on the differential regulation of both ompS1 promoters and its impact on the pathogenesis.

The nucleoid protein HU positively regulates the expression of type VI secretion systems in Enterobacter cloacae.

Enterobacter cloacae is an emerging pathogen isolated in healthcare-associated infections. A major virulence factor of this bacterium is the type VI secretion system (T6SS). The genome of E. cloacae harbors two T6SS gene clusters (T6SS-1 and T6SS-2), and the functional characterization of both systems showed that these two T6SSs are not expressed under the same conditions. Here, we report that the major histone-like protein HU positively regulates the expression of both T6SSs and, therefore, the function that each T6SS exerts in E. cloacae. Single deletions of the genes encoding the HU subunits (hupA and hupB) decreased mRNA levels of both T6SS. In contrast, the hupA hupB double mutant dramatically affected the T6SS expression, diminishing its transcription. The direct binding of HU to the promoter regions of T6SS-1 and T6SS-2 was confirmed by electrophoretic mobility shift assay. In addition, single and double mutations in the hup genes affected the ability of inter-bacterial killing, biofilm formation, adherence to epithelial cells, and intestinal colonization, but these phenotypes were restored when such mutants were trans-complemented. Our data broaden our understanding of the regulation of HU-mediated T6SS in these pathogenic bacteria. IMPORTANCE: T6SS is a nanomachine that functions as a weapon of bacterial destruction crucial for successful colonization in a specific niche. Enterobacter cloacae expresses two T6SSs required for bacterial competition, adherence, biofilm formation, and intestinal colonization. Expression of T6SS genes in pathogenic bacteria is controlled by multiple regulatory systems, including two-component systems, global regulators, and nucleoid proteins. Here, we reported that the HU nucleoid protein directly activates both T6SSs in E. cloacae, affecting the T6SS-related phenotypes. Our data describe HU as a new regulator involved in the transcriptional regulation of T6SS and its impact on E. cloacae pathogenesis.

Dissection of the role of pili and type 2 and 3 secretion systems in adherence and biofilm formation of an atypical enteropathogenic Escherichia coli strain.

Atypical enteropathogenic Escherichia coli (aEPEC) strains are diarrheal pathogens that lack bundle-forming pilus production but possess the virulence-associated locus of enterocyte effacement. aEPEC strain 1551-2 produces localized adherence (LA) on HeLa cells; however, its isogenic intimin (eae) mutant produces a diffuse-adherence (DA) pattern. In this study, we aimed to identify the DA-associated adhesin of the 1551-2 eae mutant. Electron microscopy of 1551-2 identified rigid rod-like pili composed of an 18-kDa protein, which was identified as the major pilin subunit of type 1 pilus (T1P) by mass spectrometry analysis. Deletion of fimA in 1551-2 affected biofilm formation but had no effect on adherence properties. Analysis of secreted proteins in supernatants of this strain identified a 150-kDa protein corresponding to SslE, a type 2 secreted protein that was recently reported to be involved in biofilm formation of rabbit and human EPEC strains. However, neither adherence nor biofilm formation was affected in a 1551-2 sslE mutant. We then investigated the role of the EspA filament associated with the type 3 secretion system (T3SS) in DA by generating a double eae espA mutant. This strain was no longer adherent, strongly suggesting that the T3SS translocon is the DA adhesin. In agreement with these results, specific anti-EspA antibodies blocked adherence of the 1551-2 eae mutant. Our data support a role for intimin in LA, for the T3SS translocon in DA, and for T1P in biofilm formation, all of which may act in concert to facilitate host intestinal colonization by aEPEC strains.

Identification of secretory immunoglobulin A antibody targets from human milk in cultured cells infected with enteropathogenic Escherichia coli (EPEC).

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to inject effectors into host cells and alter cellular physiology. The aim of the present study was to identify targets of human secretory immunoglobulin A (sIgA) antibodies from the proteins delivered by EPEC into HEp-2 cells after infection. Bacterial proteins delivered into EPEC-infected cells were obtained in sub-cellular fractions (cytoplasmic, membrane, and cytoskeleton) and probed with sIgA antibodies from human milk and analyzed by Western blotting. These sIgA antibodies reacted with Tir and EspB in the cytoplasmic and membrane fractions, and with intimin in the membrane fraction mainly. The sIgA also identified an EPEC surface-associated Heat-shock protein 70 (Hsp70) in HEp-2 cells infected with EPEC. Purified Hsp70 from EPEC was able to bind to HEp-2 cells, suggesting adhesive properties in this protein. EspC secreted to the medium reacted strongly with the sIgA antibodies. An EPEC 115 kDa protein, unrelated to the EspC protein, was detected in the cytoplasm of infected HEp-2 cells, suggesting that this is a new protein translocated by EPEC. The results suggest that there is a strong host antibody response to Tir and intimin, which are essential proteins for attaching and effacing (A/E) pathogen mediated disease.

Highly-conserved regulatory activity of the ANR family in the virulence of diarrheagenic bacteria through interaction with master and global regulators.

ANR (AraC negative regulators) are a novel class of small regulatory proteins commonly found in enteric pathogens. Aar (AggR-activated regulator), the best-characterized member of the ANR family, regulates the master transcriptional regulator of virulence AggR and the global regulator HNS in enteroaggregative Escherichia coli (EAEC) by protein-protein interactions. On the other hand, Rnr (RegA-negative regulator) is an ANR homolog identified in attaching and effacing (AE) pathogens, including Citrobacter rodentium and enteropathogenic Escherichia coli (EPEC), sharing only 25% identity with Aar. We previously found that C. rodentium lacking Rnr exhibits prolonged shedding and increased gut colonization in mice compared to the parental strain. To gain mechanistic insights into this phenomenon, we characterized the regulatory role of Rnr in the virulence of prototype EPEC strain E2348/69 by genetic, biochemical, and human organoid-based approaches. Accordingly, RNA-seq analysis revealed more than 500 genes differentially regulated by Rnr, including the type-3 secretion system (T3SS). The abundance of EspA and EspB in whole cells and bacterial supernatants confirmed the negative regulatory activity of Rnr on T3SS effectors. We found that besides HNS and Ler, twenty-six other transcriptional regulators were also under Rnr control. Most importantly, the deletion of aar in EAEC or rnr in EPEC increases the adherence of these pathogens to human intestinal organoids. In contrast, the overexpression of ANR drastically reduces bacterial adherence and the formation of AE lesions in the intestine. Our study suggests a conserved regulatory mechanism and a central role of ANR in modulating intestinal colonization by these enteropathogens despite the fact that EAEC and EPEC evolved with utterly different virulence programs.

Transcriptional regulation of the ecp operon by EcpR, IHF, and H-NS in attaching and effacing Escherichia coli.

Enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli are clinically important diarrheagenic pathogens that adhere to the intestinal epithelial surface. The E. coli common pili (ECP), or meningitis-associated and temperature-regulated (MAT) fimbriae, are ubiquitous among both commensal and pathogenic E. coli strains and play a role as colonization factors by promoting the interaction between bacteria and host epithelial cells and favoring interbacterial interactions in biofilm communities. The first gene of the ecp operon encodes EcpR (also known as MatA), a proposed regulatory protein containing a LuxR-like C-terminal helix-turn-helix (HTH) DNA-binding motif. In this work, we analyzed the transcriptional regulation of the ecp genes and the role of EcpR as a transcriptional regulator. EHEC and EPEC ecpR mutants produce less ECP, while plasmids expressing EcpR increase considerably the expression of EcpA and production of ECP. The ecp genes are transcribed as an operon from a promoter located 121 bp upstream of the start codon of ecpR. EcpR positively regulates this promoter by binding to two TTCCT boxes distantly located upstream of the ecp promoter, thus enhancing expression of downstream ecp genes, leading to ECP production. EcpR mutants in the putative HTH DNA-binding domain are no longer able to activate ecp expression or bind to the TTCCT boxes. EcpR-mediated activation is aided by integration host factor (IHF), which is essential for counteracting the repression exerted by histone-like nucleoid-structuring protein (H-NS) on the ecp promoter. This work demonstrates evidence about the interplay between a novel member of a diverse family of regulatory proteins and global regulators in the regulation of a fimbrial operon.

Role of curli and cellulose expression in adherence of Escherichia coli O157:H7 to spinach leaves.

Shiga-toxigenic Escherichia coli O157:H7 outbreaks have been linked to consumption of fresh produce. It is generally recognized that bacterial attachment to vegetal matrices constitutes the first step in contamination of fresh produce. Cellular appendages, such as curli fibers, and cellulose, a constituent of extracellular matrix, have been suggested to be involved in E. coli attachment and persistence in fresh produce. A comparative evaluation was conducted on the ability of Shiga toxin-producing E. coli O157:H7 strains EDL933 and 86-24, linked to two independent foodborne disease outbreaks in humans, and their mutants deficient in curli and/or cellulose expression to colonize and to firmly attach to spinach leaf. Inoculated spinach leaves were incubated at 22°C, and at 0, 24, and 48 h after incubation loosely and strongly attached E. coli O157:H7 populations were determined. Curli-expressing E. coli O157:H7 strains developed stronger association with leaf surface, whereas curli-deficient mutants attached to spinach at significantly (p<0.01) lower numbers. Attachment of cellulose-impaired mutants to spinach leaves was not significantly different from that of curliated strains. The relative attachment strength of E. coli O157:H7 to spinach increased with incubation time for the curli-expressing strains. Laser scanning confocal microscopy (LSCM) analysis of inoculated leaves revealed that curli-expressing E. coli O157:H7 were surrounded by extracellular structures strongly immunostained with anti-curli antibodies. Production of cellulose was not required to develop strong attachment to spinach leaf. These results indicate that curli fibers are essential for strong attachment of E. coli O157:H7 to spinach whereas cellulose is dispensable.

The Transcription of Flagella of Enteropathogenic Escherichia coli O127:H6 Is Activated in Response to Environmental and Nutritional Signals.

The flagella of enteropathogenic Escherichia coli (EPEC) O127:H6 E2348/69 mediate adherence to host proteins and epithelial cells. What environmental and nutritional signals trigger or down-regulate flagella expression in EPEC are largely unknown. In this study, we analyzed the influence of pH, oxygen tension, cationic and anionic salts (including bile salt), carbon and nitrogen sources, and catecholamines on the expression of the flagellin gene (fliC) of E2348/69. We found that sodium bicarbonate, which has been shown to induce the expression of type III secretion effectors, down-regulated flagella expression, explaining why E2348/69 shows reduced motility and flagellation when growing in Dulbecco's Minimal Essential Medium (DMEM). Further, growth under a 5% carbon dioxide atmosphere, in DMEM adjusted to pH 8.2, in M9 minimal medium supplemented with 80 mM glucose or sucrose, and in DMEM containing 150 mM sodium chloride, 0.1% sodium deoxycholate, or 30 µM epinephrine significantly enhanced fliC transcription to different levels in comparison to growth in DMEM alone. When EPEC was grown in the presence of HeLa cells or in supernatants of cultured HeLa cells, high levels (4-fold increase) of fliC transcription were detected in comparison to growth in DMEM alone. Our data suggest that nutritional and host signals that EPEC may encounter in the intestinal niche activate fliC expression in order to favor motility and host colonization.

The Fis Nucleoid Protein Negatively Regulates the Phase Variation fimS Switch of the Type 1 Pilus Operon in Enteropathogenic Escherichia coli.

In Escherichia coli the expression of type 1 pili (T1P) is determined by the site-specific inversion of the fimS ON-OFF switch located immediately upstream of major fimbrial subunit gene fimA. Here we investigated the role of virulence (Ler, GrlR, and GrlA) and global regulators (H-NS, IHF, and Fis) in the regulation of the fimS switch in the human enteropathogenic E. coli (EPEC) O127:H6 strain E2348/69. This strain does not produce detectable T1P and PCR analysis of the fimS switch confirmed that it is locked in the OFF orientation. Among the regulator mutants analyzed, only the ∆fis mutant produced significantly high levels of T1P on its surface and yielded high titers of agglutination of guinea pig erythrocytes. Expression analysis of the fimA, fimB, and fimE promoters using lacZ transcriptional fusions indicated that only PfimA activity is enhanced in the absence of Fis. Collectively, these data demonstrate that Fis is a negative regulator of T1P expression in EPEC and suggest that it is required for the FimE-dependent inversion of the fimS switch from the ON-to-OFF direction. It is possible that a similar mechanism of T1P regulation exists in other intestinal and extra-intestinal pathogenic classes of E. coli.

Flagellin outer domain dimerization modulates motility in pathogenic and soil bacteria from viscous environments.

Flagellar filaments function as the propellers of the bacterial flagellum and their supercoiling is key to motility. The outer domains on the surface of the filament are non-critical for motility in many bacteria and their structures and functions are not conserved. Here, we show the atomic cryo-electron microscopy structures for flagellar filaments from enterohemorrhagic Escherichia coli O157:H7, enteropathogenic E. coli O127:H6, Achromobacter, and Sinorhizobium meliloti, where the outer domains dimerize or tetramerize to form either a sheath or a screw-like surface. These dimers are formed by 180° rotations of half of the outer domains. The outer domain sheath (ODS) plays a role in bacterial motility by stabilizing an intermediate waveform and prolonging the tumbling of E. coli cells. Bacteria with these ODS and screw-like flagellar filaments are commonly found in soil and human intestinal environments of relatively high viscosity suggesting a role for the dimerization in these environments.

The Flp type IV pilus operon of Mycobacterium tuberculosis is expressed upon interaction with macrophages and alveolar epithelial cells.

The genome of Mycobacterium tuberculosis (Mtb) harbors the genetic machinery for assembly of the Fimbrial low-molecular-weight protein (Flp) type IV pilus. Presumably, the Flp pilus is essential for pathogenesis. However, it remains unclear whether the pili genes are transcribed in culture or during infection of host cells. This study aimed to shed light on the expression of the Flp pili-assembly genes (tadZ, tadA, tadB, tadC, flp, tadE, and tadF) in Mtb growing under different growth conditions (exponential phase, stationary phase, and dormancy NRP1 and NRP2 phases induced by hypoxia), during biofilm formation, and in contact with macrophages and alveolar epithelial cells. We found that expression of tad/flp genes was significantly higher in the stationary phase than in exponential or NRP1 or NRP2 phases suggesting that the bacteria do not require type IV pili during dormancy. Elevated gene expression levels were recorded when the bacilli were in contact for 4 h with macrophages or epithelial cells, compared to mycobacteria propagated alone in the cultured medium. An antibody raised against a 12-mer peptide derived from the Flp pilin subunit detected the presence of Flp pili on intra- and extracellular bacteria infecting eukaryotic cells. Altogether, these are compelling data showing that the Flp pili genes are expressed during the interaction of Mtb with host cells and highlight a role for Flp pili in colonization and invasion of the host, subsequently promoting bacterial survival during dormancy.

Bacterial macroscopic rope-like fibers with cytopathic and adhesive properties.

We present a body of ultrastructural, biochemical, and genetic evidence that demonstrates the oligomerization of virulence-associated autotransporter proteins EspC or EspP produced by deadly human pathogens enterohemorrhagic and enteropathogenic Escherichia coli into novel macroscopic rope-like structures (>1 cm long). The rope-like structures showed high aggregation and insolubility, stability to anionic detergents and high temperature, and binding to Congo Red and thioflavin T dyes. These are properties also exhibited by human amyloidogenic proteins. These macroscopic ropes were not observed in cultures of nonpathogenic Escherichia coli or isogenic espP or espC deletion mutants of enterohemorrhagic or enteropathogenic Escherichia coli but were produced by an Escherichia coli K-12 strain carrying a plasmid expressing espP. Purified recombinant EspP monomers were able to self-assemble into macroscopic ropes upon incubation, suggesting that no other protein was required for assembly. The ropes bound to and showed cytopathic effects on cultured epithelial cells, served as a substratum for bacterial adherence and biofilm formation, and protected bacteria from antimicrobial compounds. We hypothesize that these ropes play a biologically significant role in the survival and pathogenic scheme of these organisms.