A structural basis for sustained bacterial adhesion: biomechanical properties of CFA/I pili

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal disease worldwide. Adhesion pili (or fimbriae), such as the CFA/I (colonization factor antigen I) organelles that enable ETEC to attach efficiently to the host intestinal tract epithelium, are critical virulence factors for initiation of infection. We characterized the intrinsic biomechanical properties and kinetics of individual CFA/I pili at the single-organelle level, demonstrating that weak external forces (7.5 pN) are sufficient to unwind the intact helical filament of this prototypical ETEC pilus and that it quickly regains its original structure when the force is removed. While the general relationship between exertion of force and an increase in the filament length for CFA/I pili associated with diarrheal disease is analogous to that of P pili and type 1 pili, associated with urinary tract and other infections, the biomechanical properties of these different pili differ in key quantitative details. Unique features of CFA/I pili, including the significantly lower force required for unwinding, the higher extension speed at which the pili enter a dynamic range of unwinding, and the appearance of sudden force drops during unwinding, can be attributed to morphological features of CFA/I pili including weak layer-to-layer interactions between subunits on adjacent turns of the helix and the approximately horizontal orientation of pilin subunits with respect to the filament axis. Our results indicate that ETEC CFA/I pili are flexible organelles optimized to withstand harsh motion without breaking, resulting in continued attachment to the intestinal epithelium by the pathogenic bacteria that express these pili.     

肠毒素大肠杆菌定居因子抗原基因CFA-I在痢疾杆菌中的表达

通过体外重组的方法 ,构建了包含asd基因的重组表达质粒 pZHY2 1,与福氏志贺氏菌Fwl0 1构成了宿主 载体平衡致死系统 ,Western印迹结果表明 ,在没有抗生素条件选择的情况下 ,稳定表达肠毒素大肠杆菌定居因子抗原CFA I。电镜结果显示 ,在重组菌株的菌体表面 ,表达产物能够装配成菌毛。重组菌通过口服和鼻饲免疫小鼠后 ,可以诱生CFA I的抗体 ;同时可以检测到分泌型IgA产生 ,表明重组菌可以诱导相应的粘膜免疫反应     

Structure and function of enterotoxigenic Escherichia coli fimbriae from differing assembly pathways

Pathogenic enterotoxigenic Escherichia coli (ETEC) are the major bacterial cause of diarrhea in young children in developing countries and in travelers, causing significant mortality in children. Adhesive fimbriae are a prime virulence factor for ETEC, initiating colonization of the small intestinal epithelium. Similar to other Gram‐negative bacteria, ETEC express one or more diverse fimbriae, some assembled by the chaperone‐usher pathway and others by the alternate chaperone pathway. Here, we elucidate structural and biophysical aspects and adaptations of each fimbrial type to its respective host niche. CS20 fimbriae are compared with colonization factor antigen I (CFA/I) fimbriae, which are two ETEC fimbriae assembled via different pathways, and with P‐fimbriae from uropathogenic E. coli. Many fimbriae unwind from their native helical filament to an extended linear conformation under force, thereby sustaining adhesion by reducing load at the point of contact between the bacterium and the target cell. CFA/I fimbriae require the least force to unwind, followed by CS20 fimbriae and then P‐fimbriae, which require the highest unwinding force. We conclude from our electron microscopy reconstructions, modeling and force spectroscopy data that the target niche plays a central role in the biophysical properties of fimbriae that are critical for bacterial pathophysiology.     

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream¹. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates², or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.Our mouse model of intranasal colonization is adapted from human models³ and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx^4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice⁸, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.     

The AgI/II Family Adhesin AspA Is Required for Respiratory Infection by Streptococcus pyogenes

Streptococcus pyogenes (GAS) is a human pathogen that causes pharyngitis and invasive diseases such as toxic shock syndrome and sepsis. The upper respiratory tract is the primary reservoir from which GAS can infect new hosts and cause disease. The factors involved in colonisation are incompletely known however. Previous evidence in oral streptococci has shown that the AgI/II family proteins are involved. We hypothesized that the AspA member of this family might be involved in GAS colonization. We describe a novel mouse model of GAS colonization of the nasopharynx and lower respiratory tract to elucidate these interactions. We used two clinical M serotypes expressing AspA, and their aspA gene deletant isogenic mutants in experiments using adherence assays to respiratory epithelium, macrophage phagocytosis and neutrophil killing assays and in vivo models of respiratory tract colonisation and infection. We demonstrated the requirement for AspA in colonization of the respiratory tract. AspA mutants were cleared from the respiratory tract and were deficient in adherence to epithelial cells, and susceptible to phagocytosis. Expression of AspA in the surrogate host Lactococcus lactis protected bacteria from phagocytosis. Our results suggest that AspA has an essential role in respiratory infection, and may function as a novel anti-phagocytic factor.     

Hydrophobic adsorptive and hemagglutinating properties ofEscherichia coli possessing colonization factor antigens (CFA/I or CFA/II), type 1 pili,or other pili

Hydrophobic and hemagglutinating activities of piliated enterotoxigenicEscherichia coli possessing colonization factor antigens (CFA)/I and putative CFA/II, strains with type 1 pili, and piliated strains of nonenterotoxigenicE. coli from urinary tract infections were compared. When passed through columns of hydrophobic Phenyl Sepharose in the presence of buffered ammonium sulfate, the strains with CFA adsorbed most strongly. Similarly, the CFA strains showed a tendency to autoagglutinate at a lower (NH₄)₂SO₄ concentration than the other strains studied. The degree of hydrophobicity of the strains tested is in the order CFA/I>CFA/II>type 1 pili>urinary tract strains. Rough variants ofE. coli strains were more hydrophobic than their smooth parents. Electron microscopy showed large numbers of pili on CFA strains, whereas type 1 piliated strains possessed fewer pili. CFA-negative clones possessed few or no, pili and did not adsorb to the gel. A highly piliated mutant strain (PAK/2PfS) ofPseudomonas aeruginosa bound to the Phenyl Sepharose while the poorly piliated wild-type strains did not. Strains, lost their adsorptive capacity after blending, sonication, heating, or trypsin treatment. It is concluded that the hydrophobicity of enteric organisms, as measured by hydrophobic interaction chromatography, is a function of the type and number of pili on the cell surface.     

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.     

Microbes inside—from diversity to function: the case of Akkermansia

The human intestinal tract is colonized by a myriad of microbes that have developed intimate interactions with the host. In healthy individuals, this complex ecosystem remains stable and resilient to stressors. There is significant attention on the understanding of the composition and function of this intestinal microbiota in health and disease. Current developments in metaomics and systems biology approaches allow to probe the functional potential and activity of the intestinal microbiota. However, all these approaches inherently suffer from the fact that the information on macromolecules (DNA, RNA and protein) is collected at the ecosystem level. Similarly, all physiological and other information collected from isolated strains relates to pure cultures grown in vitro or in gnotobiotic systems. It is essential to integrate these two worlds of predominantly chemistry and biology by linking the molecules to the cells. Here, we will address the integration of omics- and culture-based approaches with the complexity of the human intestinal microbiota in mind and the mucus-degrading bacteria Akkermansia spp. as a paradigm.     

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.     

Crohn's Disease-Associated Adherent-Invasive Escherichia coli Adhesion Is Enhanced by Exposure to the Ubiquitous Dietary Polysaccharide Maltodextrin

Crohn''s disease (CD) is associated with intestinal dysbiosis evidenced by an altered microbiome forming thick biofilms on the epithelium. Additionally, adherent-invasive E. coli (AIEC) strains are frequently isolated from ileal lesions of CD patients indicating a potential role for these strains in disease pathogenesis. The composition and characteristics of the host microbiome are influenced by environmental factors, particularly diet. Polysaccharides added to food as emulsifiers, stabilizers or bulking agents have been linked to bacteria-associated intestinal disorders. The escalating consumption of polysaccharides in Western diets parallels an increased incidence of CD during the latter 20^th century. In this study, the effect of a polysaccharide panel on adhesiveness of the CD-associated AIEC strain LF82 was analyzed to determine if these food additives promote disease-associated bacterial phenotypes. Maltodextrin (MDX), a polysaccharide derived from starch hydrolysis, markedly enhanced LF82 specific biofilm formation. Biofilm formation of multiple other E. coli strains was also promoted by MDX. MDX-induced E. coli biofilm formation was independent of polysaccharide chain length indicating a requirement for MDX metabolism. MDX exposure induced type I pili expression, which was required for MDX-enhanced biofilm formation. MDX also increased bacterial adhesion to human intestinal epithelial cell monolayers in a mechanism dependent on type 1 pili and independent of the cellular receptor CEACAM6, suggesting a novel mechanism of epithelial cell adhesion. Analysis of mucosa-associated bacteria from individuals with and without CD showed increased prevalence of malX, a gene essential for MDX metabolism, uniquely in the ileum of CD patients. These findings demonstrate that the ubiquitous dietary component MDX enhances E. coli adhesion and suggests a mechanism by which Western diets rich in specific polysaccharides may promote dysbiosis of gut microbes and contribute to disease susceptibility.     

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.     

Structure of CfaA Suggests a New Family of Chaperones Essential for Assembly of Class 5 Fimbriae

Adhesive pili on the surface of pathogenic bacteria comprise polymerized pilin subunits and are essential for initiation of infections. Pili assembled by the chaperone-usher pathway (CUP) require periplasmic chaperones that assist subunit folding, maintain their stability, and escort them to the site of bioassembly. Until now, CUP chaperones have been classified into two families, FGS and FGL, based on the short and long length of the subunit-interacting loops between its F1 and G1 β-strands, respectively. CfaA is the chaperone for assembly of colonization factor antigen I (CFA/I) pili of enterotoxigenic E. coli (ETEC), a cause of diarrhea in travelers and young children. Here, the crystal structure of CfaA along with sequence analyses reveals some unique structural and functional features, leading us to propose a separate family for CfaA and closely related chaperones. Phenotypic changes resulting from mutations in regions unique to this chaperone family provide insight into their function, consistent with involvement of these regions in interactions with cognate subunits and usher proteins during pilus assembly.     

Relationship between intestinal microecology and the translocation of intestinal bacteria

It is now well known that endogenous bacteria can translocate from the intestinal tract and cause many of the complicating infections seen in severely ill, hospitalized patients. Of the hundreds of bacterial species in the intestinal tract, relatively few aerobic/facultative species appear to translocate with any frequency. Van der Waaij and colleagues (1971, 1972a, 1972b) originally proposed that, by a process termed colonization resistance, strictly anaerobic bacteria prevented the intestinal overgrowth and subsequent translocation of these potentially pathogenic aerobic/facultative bacteria. Selective antimicrobial decontamination, designed to maintain colonization resistance, has been effective in reducing the incidence of infectious morbidity in high risk patients. However, the mechanisms controlling bacterial translocation remain unclear, but appear to depend on host factors, as well as on factors inherent in the microbe itself. There is both clinical and experimental evidence supporting the concept that strictly anaerobic bacteria do not readily translocate. Bacteria that are able to survive within macrophages (e.g., Salmonella species and Listeria monocytogenes) translocate easier than others, and there is recent experimental evidence that normal intestinal bacteria may translocate to the draining mesenteric lymph node within host phagocytes. There is also evidence that anaerobic bacteria translocate along with facultative species in situations associated with intestinal epithelial damage, i.e., burn trauma, oral ricinoleic acid, and acute mesenteric ischemia. In contrast, recent experimental evidence demonstrates that facultative bacteria can translocate across a histologically intact intestinal epithelium, and that the ileal absorptive cell may be at least one portal of entry prior to transport into deeper tissues. It is anticipated that further clarification of the routes and mechanisms involved in bacterial translocation will provide new insights into the treatment and prevention of a significant proportion of the infectious morbidity seen in severely ill, hospitalized patients. Antoni van Leeuwenhoek Lecture presented at the Annual Meeting of the Netherlands Society of Microbiology, Utrecht, 23 November, 1989.     

A new understanding of enteroaggregative Escherichia coli as an inflammatory pathogen

Enteroaggregative Escherichia coli (EAEC) is an important cause of endemic and epidemic diarrheal disease worldwide. Although not classically considered an inflammatory pathogen in the style of Shigella and Salmonella species, clinical data from patients suggests that inflammatory responses may play an important role during EAEC disease. However, the specific role of inflammation during EAEC pathogenesis has not been investigated in detail. To better understand how EAEC may induce inflammation, we have focused our attention on the intimate interactions between EAEC and the host epithelium and the subsequent induction of host cell signaling events leading to innate immune responses. Here, we discuss our recent findings on the signaling pathway by which EAEC promotes transepithelial migration of polymorphonuclear leukocytes (PMNs), the role of aggregative adherence fimbriae in triggering this event and the implementation of human intestinal xenografts in immunodeficient mice for studying EAEC pathogenesis in vivo. Our findings suggest that EAEC shares conserved mechanisms of inducing PMN recruitment with other intestinal pathogens, providing new insight into the potential pathological consequences of EAEC-induced inflammation.     

Intestinal Colonization Dynamics of Vibrio cholerae

To cause the diarrheal disease cholera, Vibrio cholerae must effectively colonize the small intestine. In order to do so, the bacterium needs to successfully travel through the stomach and withstand the presence of agents such as bile and antimicrobial peptides in the intestinal lumen and mucus. The bacterial cells penetrate the viscous mucus layer covering the epithelium and attach and proliferate on its surface. In this review, we discuss recent developments and known aspects of the early stages of V. cholerae intestinal colonization and highlight areas that remain to be fully understood. We propose mechanisms and postulate a model that covers some of the steps that are required in order for the bacterium to efficiently colonize the human host. A deeper understanding of the colonization dynamics of V. cholerae and other intestinal pathogens will provide us with a variety of novel targets and strategies to avoid the diseases caused by these organisms.     

ATG16L1 and pathogenesis of urinary tract infections

Autophagy is generally considered to be antipathogenic. The autophagy gene ATG16L1 has a commonly occurring mutation associated with Crohn disease (CD) and intestinal cell abnormalities. Mice hypomorphic for ATG16L1 (ATG16L1^HM) recreate specific features of CD. Our recent study shows that the same ATG16L1^HM mice that are susceptible to intestinal inflammatory disease are protected from urinary tract infections (UTI), a common and important human disease primarily caused by uropathogenic E. coli (UPEC). UPEC colonize the bladder and exhibit both luminal and intra-epithelial stages. The host responds by recruiting innate immune cells and shedding infected epithelial cells to clear infection. Despite these countermeasures, UPEC can persist within the bladder epithelium as membrane-enclosed quiescent intracellular reservoirs (QIRs) that can seed recurrent UTI. The mechanisms of persistence remain unknown. In this study, we show that ATG16L1 deficiency protects the host against acute UTI and UPEC latency. ATG16L1^HM mice clear urinary bacterial loads more rapidly and thoroughly due to ATG16L1-deficient innate immune components. Furthermore, ATG16L1^HM mice exhibit superficial urothelial cell-autonomous architectural aberrations that also result in significantly reduced QIR numbers. Our findings reveal a host-protective effect of ATG16L1 deficiency in vivo against a common pathogen.     

Diverse toxic effectors are harbored by vgrG islands for interbacterial antagonism in type VI secretion system

The type VI secretion system (T6SS) is considered as one of the key competition strategies by injecting toxic effectors for intestinal pathogens to acquire optimal colonization in host gut, a microenviroment with high-density polymicrobial community where bacteria compete for niches and resources. Enterotoxigenic Escherichia coli (ETEC), a major cause of infectious diarrhea in human and animals, widely encode T6SS clusters in their genomes. In this report, we first identified VT1, a novel amidase effector in ETEC, significantly hydrolyzed D-lactyl-L-Ala crosslinks between N-acetylmuramoyl and L-Ala in peptidoglycan. Further study showed that the VT1/VTI1 effector/immunity pair is encoded within a typical vgrG island, and plays a critical role for the successful establishment of ETEC in host gut. Numerous putative effectors with diverse toxin domains were found by retrieving vgrG islands in pathogenic E. coli, and designated as VT modules. Therein, VT5, a lysozyme-like effector widely encoded in ETEC, was confirmed to effectively kill adjacent cells, suggesting that VT toxin modules may be critical for pathogenic E. coli to seize a significantly competitive advantage for optimal intestinal colonization. To expand our analyses for large-scale search of VT antibacterial effectors based on vgrG island, >200 predicted effectors from 20 bacterial species were found and classified into 11 predicted toxins. This work reports a new retrieval strategy for screening T6SS effectors, and provides an example how pathogenic bacteria antagonize and displace commensal microbiome to successfully colonize in the host niches through a T6SS-dependent manner.     

In vitro adherence study using formalin-treated human small intestine: Fimbria-meidated adherence of enterotoxigenic Escherichia coli and Salmonella typhi oral-route vaccine strain possessing E. coli colonization factor antigen to villi and lymphoid follicle epithelium

Abstract Enterotoxigenic Escherichia coli possessing colonization factor antigen (CFA)/I or CFA/II adhered to formalin-fixed human ileal villi with their CFA/I- or CFA/II-fimbriae. This adherence was host-specific. Adherence to formalin-fixed human ileal lymphoid follicle epithelium or mucus was also observed, although to a lesser extent. Salmonella typhi oral-route live vaccine strain possessing E. coli CFA/I manifested increased adherence to the human ileal mucosa.     

Zebrafish as a Natural Host Model for Vibrio cholerae Colonization and Transmission

The human diarrheal disease cholera is caused by the aquatic bacterium Vibrio cholerae. V. cholerae in the environment is associated with several varieties of aquatic life, including insect egg masses, shellfish, and vertebrate fish. Here we describe a novel animal model for V. cholerae, the zebrafish. Pandemic V. cholerae strains specifically colonize the zebrafish intestinal tract after exposure in water with no manipulation of the animal required. Colonization occurs in close contact with the intestinal epithelium and mimics colonization observed in mammals. Zebrafish that are colonized by V. cholerae transmit the bacteria to naive fish, which then become colonized. Striking differences in colonization between V. cholerae classical and El Tor biotypes were apparent. The zebrafish natural habitat in Asia heavily overlaps areas where cholera is endemic, suggesting that zebrafish and V. cholerae evolved in close contact with each other. Thus, the zebrafish provides a natural host model for the study of V. cholerae colonization, transmission, and environmental survival.