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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.

Bacteria have developed an exclusive signal transduction system involving multiple diguanylate cyclase and phosphodiesterase domain-containing proteins (GGDEF and EAL/HD-GYP, respectively) that modulate the levels of the same diffusible molecule, 3 -5 -cyclic diguanylic acid (c-di-GMP), to transmit signals and obtain specific cellular responses. Current knowledge about c-di- GMP signaling has been inferred mainly from the analysis of recombinant bacteria that either lack or overproduce individual members of the pathway, without addressing potential compensatory effects or interferences between them. Here, we dissected c-di-GMP signaling by constructing a Salmonella strain lacking all GGDEF-domain proteins and then producing derivatives, each restoring 1 protein. Our analysis showed that most GGDEF proteins are constitutively expressed and that their expression levels are not interdependent. Complete deletion of genes encoding GGDEFdomain proteins abrogated virulence, motility, long-term survival, and cellulose and fimbriae synthesis. Separate restoration revealed that 4 proteins from Salmonella and 1 from Yersinia pestis exclusively restored cellulose synthesis in a c-di-GMP–dependent manner, indicating that c-di-GMP produced by different GGDEF proteins can activate the same target. However, the restored strain containing the STM4551-encoding gene recovered all other phenotypes by means of gene expression modulation independently of c-di-GMP. Specifically, fimbriae synthesis and virulence were recovered through regulation of csgD and the plasmid-encoded spvAB mRNA levels, respectively. This study provides evidence that the regulation of the GGDEF-domain proteins network occurs at 2 levels: a level that strictly requires c-di-GMP to control enzymatic activities directly, restricted to cellulose synthesis in our experimental conditions, and another that involves gene regulation for which c-di-GMP synthesis can be dispensable. This research was supported by Grants GEN2003–20234-C06–05 from Acción Estategica de Genómica y Proteómica, CTQ2007-68014-C02-01 from the Ministerio de Educación y Ciencia (Spain), and 2005SGR-693 from Generalitat de Catalunya. B.G. is the recipient of a postdoctoral contract under Grant GEN2003-20234.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a food-borne pathogen that causes hemorrhagic colitis and the hemolytic uremic syndrome. Colonization of the human gut mucosa and production of potent Shiga toxins are critical virulence traits of EHEC. Although EHEC O157:H7 contains numerous putative pili operons, their role in the colonization of the natural bovine or accidental human hosts remains largely unknown. We have identified in EHEC an adherence factor, herein called E. coli common pilus (ECP), composed of a 21-kDa pilin subunit whose amino acid sequence corresponds to the product of the yagZ (renamed ecpA ) gene present in all E. coli genomes sequenced to date. ECP production was demonstrated in 121 (71.6%) of a total of 169 ecpA + strains representing intestinal and extraintestinal pathogenic as well as normal flora E. coli . High-resolution ultrastructural and immunofluorescence studies demonstrated the presence of abundant peritrichous fibrillar structures emanating from the bacterial surface forming physical bridges between bacteria adhering to cultured epithelial cells. Isogenic ecpA mutants of EHEC O157:H7 or fecal commensal E. coli showed significant reduction in adherence to cultured epithelial cells. Our data suggest that ECP production is a common feature of E. coli colonizing the human gut or other host tissues. ECP is a pilus of EHEC O157:H7 with a potential role in host epithelial cell colonization and may represent a mechanism of adherence of both pathogenic and commensal E. coli .

Statherin is an enamel pellicle protein that inhibits hydroxyapatite (HAP) nucleation and growth, lubricates the enamel surface, and is recognized by oral bacteria in periodontal diseases. We report here from solid-state NMR measurements that the protein's C-terminal region folds into an α-helix upon adsorption to HAP crystals. This region contains the binding sites for bacterial fimbriae that mediate bacterial cell adhesion to the surface of the tooth. The helical segment is shown through long-range distance measurements to fold back onto the intermediate region (residues Y16–P28) defining the global fold of the protein. Statherin, previously shown to be unstructured in solution, undergoes conformation selection on its substrate mineral surface. This surface-induced folding of statherin can be related to its functionality in inhibiting HAP crystal growth and can explain how oral pathogens selectively recognize HAP-bound statherin.

To successfully propagate and cause disease, pathogenic bacteria must modulate their transcriptional activities in response to pressures exerted by the host immune system, including secreted immunoglobulins such as secretory IgA (S-IgA), which can bind and agglutinate bacteria. Here, we present a previously undescribed flow cytometry-based screening method to identify bacterial genes expressedin vitroand repressed during infections ofVibrio cholerae, an aquatic Gram-negative bacterium responsible for the severe diarrheal disease cholera. We identified a type IV mannose-sensitive hemagglutinin (MSHA) pilus that is repressed specificallyin vivo. We showed that bacteria that failed to turn off MSHA biosynthesis were unable to colonize the intestines of infant mice in the presence of S-IgA. We also found thatV. choleraebound S-IgA in an MSHA-dependent and mannose-sensitive fashion and that binding of S-IgA prevented bacteria from penetrating mucus barriers and attaching to the surface of epithelial cells. The ability ofV. choleraeto evade the non-antigen-specific binding of S-IgA by down-regulating a surface adhesin represents a previously undescribed mechanism of immune evasion in pathogenic bacteria. In addition, we found that repression of MSHA was mediated by the key virulence transcription factor ToxT, indicating thatV. choleraeis able to coordinate both virulence gene activation and repression to evade host defenses and successfully colonize intestines.

Myxococcus xanthus is a Gram-negative bacterium with a complex life cycle that includes vegetative swarming and fruiting-body formation. Social (S)-motility (coordinated movement of large cell groups) requires both type IV pili and fibrils (extracellular matrix material consisting of polysaccharides and protein). Little is known about the role of this extracellular matrix, or fibril material, in pilus-dependent motility. In this study, mutants lacking fibril material and, therefore, S-motility were found to be hyperpiliated. We demonstrated that addition of fibril material resulted in pilus retraction and rescued this phenotype. The fibril material was further examined to determine the component(s) that were responsible for triggering pilus retraction. Protein-free fibril material was found to be highly active in correcting hyperpiliation. However, the amine sugars present in hydrolyzed fibril material, e.g., glucosamine and N -acetylglucosamine (GlcNAc) had no effect on fibril − mutants, but, interestingly, cause hyperpiliation in wild-type cells. In contrast, chitin, a natural GlcNAc polymer, was found to restore pilus retraction in hyperpiliated mutants, indicating that a polysaccharide containing amine sugars is likely required for pilus retraction. These data suggest that the interaction of type IV pili with amine-containing polysaccharides on cell and slime-trail surfaces may trigger pilus retraction, resulting in S-motility and slime-trailing behaviors.

Type IV pili are thin filaments that extend from the poles of a diverse group of bacteria, enabling them to move at speeds of a few tenths of a micrometer per second. They are required for twitching motility, e.g., in Pseudomonas aeruginosa and Neisseria gonorrhoeae , and for social gliding motility in Myxococcus xanthus . Here we report direct observation of extension and retraction of type IV pili in P. aeruginosa . Cells without flagellar filaments were labeled with an amino-specific Cy3 fluorescent dye and were visualized on a quartz slide by total internal reflection microscopy. When pili were attached to a cell and their distal ends were free, they extended or retracted at rates of about 0.5 μm s −1 (29°C). They also flexed by Brownian motion, exhibiting a persistence length of about 5 μm. Frequently, the distal tip of a filament adsorbed to the substratum and the filament was pulled taut. From the absence of lateral deflections of such filaments, we estimate tensions of at least 10 pN. Occasionally, cell bodies came free and were pulled forward by pilus retraction. Thus, type IV pili are linear actuators that extend, attach at their distal tips, exert substantial force, and retract.

Haemophilus influenzae is a Gram-negative bacterium that represents a common cause of human disease. Disease due to this organism begins with colonization of the upper respiratory mucosa, a process facilitated by adhesive fibers called pili. In the present study, we investigated the structure and assembly of H. influenzae pili. Examination of pili by electron microscopy using quick-freeze, deep-etch and immunogold techniques revealed the presence of two distinct subassemblies, including a flexible two-stranded helical rod comprised of HifA and a short, thin, distal tip structure containing HifD. Genetic and biochemical studies demonstrated that the biogenesis of H. influenzae pili is dependent on a periplasmic chaperone called HifB, which belongs to the PapD family of immunoglobulin-like chaperones. HifB bound directly to HifA and HifD, forming HifB-HifA and HifB-HifD complexes, which were purified from periplasmic extracts by ion-exchange chromatography. Continued investigation of the biogenesis of H. influenzae pili should provide general insights into organelle development and may suggest novel strategies for disease prevention.

Urinary tract infections, caused mainly by Escherichia coli, are among the most common infectious diseases. Most isolates of the uropathogenic E.coli can express type 1 and P fimbriae containing adhesins that recognize cell receptors. While P fimbriae recognize kidney glycolipid receptors and are involved in peyelonephritis, the urothelial for type 1 fimbriae were not identified. We show that type 1-fimbriated E. coli recognize uroplakins Ia and Ib, two major glycoproteins of urothelial apical plaques. Anchorage of E. coli to urothelial surface via type 1 fimbriae-uroplakin I interactions may play a role in its bladder colonization and eventual ascent through the ureters, against urine flow, to invade the kidneys.

Type 1 fimbriae are adhesion organelles expressed by many Gram-negative bacteria. They facilitate adherence to mucosal surfaces and inflammatory cells in vitro, but their contribution to virulence has not been defined. This study presents evidence that type 1 fimbriae increase the virulence of Escherichia coli for the urinary tract by promoting bacterial persistence and enhancing the inflammatory response to infection. In a clinical study, we observed that disease severity was greater in children infected with E. coli O1:K1:H7 isolates expressing type 1 fimbriae than in those infected with type 1 negative isolates of the same serotype. The E. coli O1:K1:H7 isolates had the same electrophoretic type, were hemolysin-negative, expressed P fimbriae, and carried the fim DNA sequences. When tested in a mouse urinary tract infection model, the type 1-positive E. coli O1:K1:H7 isolates survived in higher numbers, and induced a greater neutrophil influx into the urine, than O1:K1:H7 type 1-negative isolates. To confirm a role of type 1 fimbriae, a fimH null mutant (CN1016) was constructed from an O1:K1:H7 type 1-positive parent. E. coli CN1016 had reduced survival and inflammatogenicity in the mouse urinary tract infection model. E. coli CN1016 reconstituted with type 1 fimbriae (E. coli CN1018) had restored virulence similar to that of the wild-type parent strain. These results show that type 1 fimbriae in the genetic background of a uropathogenic strain contribute to the pathogenesis of E. coli in the urinary tract.

We investigated the role of the Salmonella typhimurium fimbrial operon formed by the genes lpfABCDE in infection of mice. A mutant in lpfC, the gene encoding the fimbrial outer membrane usher, had an approximately 5-fold increased 50% lethal dose when administered orally to mice. When mice were infected with a mixture of the lpfC mutant and isogenic wild-type S. typhimurium, the lpfC mutant was recovered in lower numbers from Peyer's patches, mesenteric lymph nodes, liver, and spleen. In an organ culture model using murine intestinal loops, lpfC mutants were shown to be associated in lower numbers than wild-type bacteria with Peyer's patches but not with villous intestine. The defect of the lpfC mutant in adhesion to Peyer's patches could be complemented by introducing lpfABCDE on a cosmid. Similarly, heterologous expression of the Salmonella lpf operon in Escherichia coli resulted in an increased adhesion to histological thin sections of Peyer's patch lymph follicles. Electron microscopic analysis of histological sections taken from Peyer's patches after intragastric infection of mice showed that, in contrast to the S. typhimurium wild type, the isogenic lpfC mutant did not destroy M cells of the follicle-associated epithelium. These data show that the Salmonella lpf operon is involved in adhesion to murine Peyer's patches.

The expression of type 1 fimbriae (pili) of Escherichia coli is turned on and off at the transcriptional level at a high frequency (10(-3) per cell per generation) in a process termed phase variation. Using Southern blot and DNA sequence analysis, we have detected a genomic rearrangement in the switch region immediately upstream of the fimbrial structural gene. This rearrangement involves an invertible 314-base-pair segment of DNA whose alternating orientation apparently results in the on-and-off activation of a promoter that determines the state of fimbrial expression.

Two basic alpha-helical plans appear to characterize portions of the proteins projecting from the surface of many microorganisms. Structure A is related to that of the antibody-binding protein A of Staphylococcus aureus, in which a single polypeptide chain doubles back to form an Anti-parallel domain; the other, structure M, is related to the M proteins of group A streptococci, in which Multiple parallel alpha-helical chains interlock in a coiled-coil conformation. A projection about 100 A long may be organized on either plan, but very long projections are likely to be of the M pattern. The alpha-helical motif may account for the length of these spikes, with other functions such as anchoring or antiphagocytic activity located in domains of different structure.