All cells have stress response pathways that maintain homeostasis in each cellular compartment. In the Gram-negative bacterium Escherichia coli, the E pathway responds to protein misfolding in the envelope. The stress signal is transduced across the inner membrane to the cytoplasm via the inner membrane protein RseA, the anti-sigma factor that inhibits the transcriptional activity of E . Stress-induced activation of the pathway requires the regulated proteolysis of RseA. In this report we show that RseA is degraded by sequential proteolytic events controlled by the inner membrane-anchored protease DegS and the membrane-embedded metalloprotease YaeL, an ortholog of mammalian Site-2 protease (S2P). This is consistent with the mechanism of activation of ATF6, the mammalian unfolded protein response transcription factor by Site-1 protease and S2P. Thus, mammalian and bacterial cells employ a conserved proteolytic mechanism to activate membrane-associated transcription factors that initiate intercompartmental cellular stress responses.

We identified the thiomuracins, a novel family of thiopeptides produced by a rare-actinomycete bacterium typed as a Nonomuraea species, via a screen for inhibition of growth of the bacterial pathogen Staphylococcus aureus. Thiopeptides are a class of macrocyclic, highly modified peptides that are decorated by thiazoles and defined by a central six-membered heterocyclic ring system. Mining the genomes of thiopeptide-producing strains revealed the elusive biosynthetic route for this class of antibiotics. The thiopeptides are chromosomally encoded, ribosomally synthesized proteins, and isolation of gene clusters for production of thiomuracin and the related thiopeptide GE2270A revealed the post-translational machinery required for maturation. The target of the thiomuracins was identified as bacterial Elongation Factor Tu (EF-Tu). In addition to potently inhibiting a target that is unexploited by marketed human therapeutics, the thiomuracins have a low propensity for selecting for antibiotic resistance and confer no measurable cross-resistance to antibiotics in clinical use.

Combinatorial libraries of rearranged hypervariable V(H) and V(L) sequences from nonimmunized human donors contain antigen specificities, including anti-self reactivities, created by random pairing of V(H)s and V(L)s. Somatic hypermutation of immunoglobulin genes, however, is critical in the generation of high-affinity antibodies in vivo and occurs only after immunization. Thus, in combinatorial phage display libraries from nonimmunized donors, high-affinity antibodies are rarely found. Lengthy in vitro affinity maturation is often needed to improve antibodies from such libraries. We report the construction of human Fab libraries having a unique combination of immunoglobulin sequences captured from human donors and synthetic diversity in key antigen contact sites in heavy-chain complementarity-determining regions 1 and 2. The success of this strategy is demonstrated by identifying many monovalent Fabs against multiple therapeutic targets that show higher affinities than approved therapeutic antibodies. This very often circumvents the need for affinity maturation, accelerating discovery of antibody drug candidates.

The evolution of drug resistance mechanisms in pathogenic microorganisms poses a global health crisis, and millions of people are at risk if the problem is not addressed in the laboratory and translated to the clinic. What role do diagnostics have in managing the challenge of antimicrobial resistance?Antibiotic resistance has emerged as a global health crisis and if we cannot reverse the trend we are on a course towards a post-antibiotic era. There are several ways that diagnostic testing can assist in managing this challenge.At a very high level, rapid near-patient diagnostic assays to distinguish viral from bacterial infections may prevent treatment with unnecessary antibiotics. If a bacterial infection is present, defining the aetiology and antibiotic susceptibility profile is essential to optimize and narrow antimicrobial therapy as quickly as possible. Whereas traditional methods used in clinical microbiology laboratories would typically require 48 h (or longer) for definitive results, rapid diagnostic methods are becoming available for routine clinical use; some of these methods can provide results within hours. Although rapid tests for infection of antimicrobial resistance, as it limits the exposure of patients and environments to antibacterials under only those circumstances in which the drugs are likely to improve the health status of an individual. The more inappropriately antibacterials are used, the more we risk the erosion of the utility of a drug due to resistance. With the immense amount of time and resources that it takes to discover new safe and effective antibiotics, diagnostics help the medical community to preserve the utility of these precious drugs.Diagnostic techniques may contribute to the identification of rapidly emerging resistance traits. First, such a rapid identification can contribute to better antibiotic stewardship. Rapid adaptation of the antibiotic therapy to the resistance phenotype of the infecting organism may save the lives of patients, as it has been shown that the optimization of the antibiotic therapy during the first 6-12 h of infection is crucial for the treatment of life-threatening infections. This is particularly true for infections caused by Gram-negative bacteria, such as those species that are currently the main focus of antibiotic resistance research. For example, species in the Enterobacteriaceae family that may produce extended-spectrum β-lactamases that confer resistance to extended-spectrum cephalosporins, carbapenemases that confer resistance to carbapenems (imipenem, meropenem or ertapenem), or that are resistant to polymyxins. Second, the rapid identification of resistance traits may contribute to the identification of patients who are infected with resistant pathogens but do not show symptoms, particularly in hospital settings. In turn, the immediate isolation of an infected individual in healthcare facilities could prevent the development of outbreaks that are associated with multidrug-resistant bacteria and save costs.Rapid and comprehensive diagnostic...

Escherichia coli hlyCABD operons encode the polypeptide component (HlyA) of an extracellular cytolytic toxin, as well as proteins required for its acylation (HlyC) and sec-independent secretion (HlyBD). Previous reports suggested that the E. coli protein RfaH is required for wild-type hemolysin expression, either by positively activating hly transcript initiation (M. J. A. Bailey, V. Koronakis, T. Schmoll, and C. Hughes, Mol. Microbiol. 6:1003-1012, 1992) or by promoting proper insertion of hemolysin export machinery in the E. coli outer membrane (C. Wandersman and S. Letoffe, Mol. Microbiol. 7:141-150, 1993). RfaH is also required for wild-type levels of mRNA transcribed from promoter-distal genes in the rfaQ-K, traY-Z, and rplK-rpoC gene clusters, suggesting that RfaH is a transcriptional antiterminator. We tested these models by analyzing the effects of rfaH mutations on hlyCABD mRNA synthesis and decay, HlyA protein levels, and hemolytic activity. The model system included a uropathogenic strain of E. coli harboring hlyCABD on the chromosome and E. coli K-12 transformed with the hlyCABD operon on a recombinant plasmid. Our results suggest that RfaH enhances hlyCABD transcript elongation, consistent with the model of RfaH involvement in transcriptional antitermination in E. coli. We also demonstrated that RfaH increases toxin efficacy. Modulation of hemolysin activity may be an indirect effect of RfaH-dependent E. coli outer membrane chemotype, which is consistent with the model of lipopolysaccharide involvement in hemolytic activity.Synthesis of Escherichia coli hemolysin is directed from operons consisting of four contiguous genes, hlyCABD. Hemolysin determinants are found on large, transmissible plasmids in animal isolates of E. coli or within unique chromosomal inserts, absent in E. coli K-12, of human uropathogenic E. coli isolates (22, 35). The hlyA gene encodes the polypeptide component of hemolysin, which undergoes HlyC-dependent acylation (15, 23) and is then transported across both bacterial membranes without an amino-terminal cleavage or a periplasmic intermediate (12,13). Hemolysin transport requires HlyB and HlyD (32), as well as the unlinked tolC gene product (33). The coding regions of individual hemolysin determinants display 97% sequence identity (16); however, untranslated regions and sequences 5Ј to the promoters are heterologous (9,16,19,20,31,36). This suggests that effectors of hemolysin transcript initiation differ among individual hlyCABD determinants, whereas regulatory features common to hemolysin operons, such as the putative rho-independent hlyA-B intergenic transcriptional terminator, may exist within the conserved transcribed regions (13).Recently, a trans-acting factor affecting the expression of several hemolysin determinants of chromosomal or plasmid origin was identified independently by two groups of investigators. The rfaH gene product was described by Bailey et al. (1) as a positive activator of hlyA transcription and by Wandersman and Letoffe (34) as a product required fo...