Eculizumab, a monoclonal antibody (mAb) directed against complement protein C5, is considered to be the current standard of care for patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome. This study describes the generation and preclinical attributes of ALXN1210, a new long-acting anti-C5 mAb, obtained through select modifications to eculizumab to both largely abolish target-mediated drug disposition (TMDD) and increase recycling efficiency via the neonatal Fc receptor (FcRn). To attenuate the effect of TMDD on plasma terminal half-life (t1/2), histidine substitutions were engineered into the complementarity-determining regions of eculizumab to enhance the dissociation rate of the mAb:C5 complex in the acidic early endosome relative to the slightly basic pH of blood. Antibody variants with optimal pH-dependent binding to C5 exhibited little to no TMDD in mice in the presence of human C5. To further enhance the efficiency of FcRn-mediated recycling of the antibody, two additional substitutions were introduced to increase affinity for human FcRn. These substitutions yielded an additional doubling of the t½ of surrogate anti-mouse C5 antibodies with reduced TMDD in transgenic mice expressing the human FcRn. In conclusion, ALXN1210 is a promising new therapeutic candidate currently in clinical development for treatment of patients with PNH and atypical hemolytic uremic syndrome.

The biology of Escherichia coli in its primary niche, the animal intestinal tract, is remarkably unexplored. Studies with the streptomycin-treated mouse model have produced important insights into the metabolic requirements for Escherichia coli to colonize mice. However, we still know relatively little about the physiology of this bacterium growing in the complex environment of an intestine that is permissive for the growth of competing flora. We have developed a system for studying colonization using an E. coli strain, MP1, isolated from a mouse. MP1 is genetically tractable and does not require continuous antibiotic treatment for stable colonization. As an application of this system, we separately knocked out each two-component system response regulator in MP1 and performed competitions against the wild-type strain. We found that only three response regulators, ArcA, CpxR, and RcsB, produce strong colonization defects, suggesting that in addition to anaerobiosis, adaptation to cell envelope stress is a critical requirement for E. coli colonization of the mouse intestine. We also show that the response regulator OmpR, which had previously been hypothesized to be important for adaptation between in vivo and ex vivo environments, is not required for MP1 colonization due to the presence of a third major porin. Escherichia coli is one of the most extensively studied and bestcharacterized organisms. Its high growth rate, facile genetics, and simple nutritional requirements have made this bacterium an excellent model system for studying basic aspects of molecular biology and bacteriology and the primary host for DNA and protein engineering. The physiology of E. coli growth and survival under diverse conditions has been intensively studied, and a significant fraction of E. coli gene products and regulatory networks have been characterized. However, for such a well-studied organism, we know remarkably little about the biology of E. coli in its primary niche: the animal gastrointestinal tract.E. coli is generally the most abundant aerobe in the intestines of warm-blooded vertebrates, although its numbers vary considerably with animal host and geography (1-3). As a species, this bacterium has a remarkable genetic diversity; the number of genes in common among fully sequenced isolates is less than half the number of genes in any individual strain (4-6). Some E. coli strains are pathogenic, depending on the host and site of infection (3, 7-9), and have been intensively studied to understand the factors controlling their virulence. However, the majority of E. coli strains associated with animals are believed to be part of the normal flora of the intestine, growing asymptomatically as commensals.Most of our knowledge about E. coli colonization of the animal intestine comes from studies with streptomycin-resistant strains colonizing mice fed streptomycin continuously in their drinking water (10, 11). This streptomycin-treated mouse model has played a key role in the characterization of the growth of E. coli in the intestine a...

Bacterial biofilm formation is thought to enhance survival in natural environments and during interaction with hosts. A robust colonizer of the human gastrointestinal tract, Escherichia coli Nissle 1917, is widely employed in probiotic therapy. In this study, we performed a genetic screen to identify genes that are involved in Nissle biofilm formation. We found that F1C fimbriae are required for biofilm formation on an inert surface. In addition, these structures are also important for adherence to epithelial cells and persistence in infant mouse colonization. The data suggest a possible connection between Nissle biofilm formation and the survival of this commensal within the host. Further study of the requirements for robust biofilm formation may improve the therapeutic efficacy of Nissle 1917.

Yersinia pestis, the causative agent of plague, expresses the Psa fimbriae (pH 6 antigen) in vitro and in vivo. To evaluate the potential virulence properties of Psa for pneumonic plague, an Escherichia coli strain expressing Psa was engineered and shown to adhere to three types of human respiratory tract epithelial cells. Psa binding specificity was confirmed with Psa-coated polystyrene beads and by inhibition assays. Individual Y. pestis cells were found to be able to express the capsular antigen fraction 1 (F1) concomitantly with Psa on their surface when analyzed by flow cytometry. To better evaluate the separate effects of F1 and Psa on the adhesive and invasive properties of Y. pestis, isogenic ⌬caf (F1 genes), ⌬psa, and ⌬caf ⌬psa mutants were constructed and studied with the three respiratory tract epithelial cells. The ⌬psa mutant bound significantly less to all three epithelial cells compared to the parental wild-type strain and the ⌬caf and ⌬caf ⌬psa mutants, indicating that Psa acts as an adhesin for respiratory tract epithelial cells. An antiadhesive effect of F1 was clearly detectable only in the absence of Psa, underlining the dominance of the Psa ؉ phenotype. Both F1 and Psa inhibited the intracellular uptake of Y. pestis. Thus, F1 inhibits bacterial uptake by inhibiting bacterial adhesion to epithelial cells, whereas Psa seems to block bacterial uptake by interacting with a host receptor that doesn't direct internalization. The ⌬caf ⌬psa double mutant bound and invaded all three epithelial cell types well, revealing the presence of an undefined adhesin(s) and invasin(s).Since the last plague pandemic at the end of the 19th century, its bacterial agent, Yersinia pestis, has been maintained in rodents in several Asian, African, and American countries, including the United States (17, 40). Bubonic plague results from the transmission of Y. pestis by flea bites. In contrast, primary pneumonic plague is acquired when a mammalian host inhales particles or aerosols carrying Y. pestis. Although plague is currently not a major public health problem in developed countries and has been suggested to be less contagious than commonly believed (32), the spread of Y. pestis by aerosols could cause a cluster of human cases of primary pneumonic plague with potential amplification of the outbreak (27).The major adhesins and invasins of enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica (YadA, Ail, and Inv) are not expressed by Y. pestis strains (15,45,51). Thus, how Y. pestis attaches to and translocates through the epithelial layer of the respiratory tract to reach deeper tissues and the bloodstream following airborne transmission remains unknown. Interestingly, Y. pestis exhibits an extensive extracellular lifestyle due to the intracellular delivery of several antiphagocytic effector proteins by its type III secretion system (T3SS-1 or Yops regulon) (5-7, 14, 54, 56). Moreover, two antigenic surface structures exported by usher-chaperone proteins characteristic of fimbrial biogenesis systems...

The natural diversity of the elt operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Enterotoxigenic Escherichia coli (ETEC)-associated diarrhea represents a major cause of mortality and morbidity among children and travelers, respectively, in most developing countries in Latin America, Africa, and Asia (3, 33). ETEC secretory diarrhea involves a rather straightforward pathogenesis plan, requiring first colonization of small intestine epithelial cells by means of filamentous adhesins collectively known as colonization factors (CFs) and, at a second stage, production of at least one out of two enterotoxin types, the heat-stable toxin (ST) and/or the heat-labile toxin (LT) (28, 36). One of the most complex aspects of ETEC pathogenesis is the remarkable antigen heterogeneity. At least 150 O:H serotypes have been found among ETEC strains isolated from humans, although a more restricted number of serotype combinations is detected among strains isolated from patients requiring medical intervention, also characterized, in some cases, by a conserved set of virulence-associated factors and a common clonal origin (29,30,46). Moreover, the ETEC phenotypic heterogeneity is also well illustrated by the encoded virulence-associated factors, including more than 20 known CFs and production of LT, ST, or both enterotoxins (10,33,46). Two types of ST, STa and STb (also known as ST-I and ST-II), have been differentiated based on biological and chemical features (7,11). Similarly, LTs produced by ETEC strains are also a heterogeneous group of toxins. Two major LT families have been identified, LT-I and LT-II. LT-II is rarely found among human-derived ETEC strains, but two natural variants have been reported, LT-IIa and LT-IIb, based on differences in the subunit sequences (14, 16). LT-I shows a rather high similarity with cholera toxin (CT) (over 80% amino acid identity), and both have been intensively studied as virulence factors and modulators of immune responses in mammalian species, including humans (18,28).The known natural variability of LT-I toxins expressed by ETEC strains has been mainly restricted to the differences detected between LTs produced by human (LTh)-and pig (LTp)-derived strains. Initial evidence based on the antigenicities and electrophoretic mobilities of LTh and LTp indicated that the toxins differ in their primary amino acid sequences (19,42). Sequencing of the elt operons encoding LTh and LTp revealed differences in the primary sequences of the toxins, which share over 95% identity along the complete amino acid sequence (45). Altogether, six amino acid replacements were detected between the A subunits (K4R, K213E, and N238D) and the B subunits (S4T, A46E, and E102K) of LTh and LTp derived from th...