Mechanism of action of and mechanism of reduced susceptibility to the novel anti-Clostridium difficile compound LFF571.

LFF571 is a novel semisynthetic thiopeptide and potent inhibitor of Gram-positive bacteria. We report that the antibacterial activity of LFF571 against Clostridium difficile is due to inhibition of translation. Single-step mutants of C. difficile with reduced susceptibility to LFF571 were selected at frequencies of <4.5 × 10(-11) to 1.2 × 10(-9). Sequencing revealed a G260E substitution in the thiopeptide-binding pocket of elongation factor Tu. Importantly, this mutation did not confer cross-resistance to clinically used antimicrobials. These results support the development of LFF571 as a treatment for C. difficile infection.

In vitro and in vivo activities of novel, semisynthetic thiopeptide inhibitors of bacterial elongation factor Tu.

Recently, we identified aminothiazole derivatives of GE2270 A. These novel semisynthetic congeners, like GE2270 A, target the essential bacterial protein elongation factor Tu (EF-Tu). Medicinal chemistry optimization of lead molecules led to the identification of preclinical development candidates 1 and 2. These cycloalklycarboxylic acid derivatives show activity against difficult to treat Gram-positive pathogens and demonstrate increased aqueous solubility compared to GE2270 A. We describe here the in vitro and in vivo activities of compounds 1 and 2 compared to marketed antibiotics. Compounds 1 and 2 were potent against clinical isolates of methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci (MIC(90) ≤ 0.25 µg/ml) but weaker against the streptococci (MIC(90) ≥ 4 µg/ml). Like GE2270 A, the derivatives inhibited bacterial protein synthesis and selected for spontaneous loss of susceptibility via mutations in the tuf gene, encoding EF-Tu. The mutants were not cross-resistant to other antibiotic classes. In a mouse systemic infection model, compounds 1 and 2 protected mice from lethal S. aureus infections with 50% effective doses (ED(50)) of 5.2 and 4.3 mg/kg, respectively. Similarly, compounds 1 and 2 protected mice from lethal systemic E. faecalis infections with ED(50) of 0.56 and 0.23 mg/kg, respectively. In summary, compounds 1 and 2 are active in vitro and in vivo activity against difficult-to-treat Gram-positive bacterial infections and represent a promising new class of antibacterials for use in human therapy.

The transmembrane domain of the DnaJ-like protein DjlA is a dimerisation domain.

DjlA is a bitopic inner membrane protein, which belongs to the DnaJ co-chaperone family in Escherichia coli. Overproduction of DjlA leads to the synthesis of colanic acid, resulting in mucoidy, via the activation of the two-component regulatory system RcsC/B that controls the cps (capsular polysaccharide) operon. This induction requires both the co-chaperone activity of DjlA, in cooperation with DnaK and GrpE, and its unique transmembrane (TM) domain. Here, we show that the TM segment of DjlA acts as a dimerisation domain: when fused to the N-terminal DNA-binding domain of the lambda cI repressor protein, it can substitute for the native C-terminal dimerisation domain of cI, thus generating an active cI repressor. Replacing the TM domain of DjlA by other TM domains, with or without dimerising capacity, revealed that dimerisation is not sufficient for the induction of cps expression, indicating an additional sequence- or structurally specific role for the TM domain. Finally, the conserved glycines present in the TM domain of DjlA are essential for the induction of mucoidy, but not for dimerisation.

Genetic selection for and molecular dynamic modeling of a protein transmembrane domain multimerization motif from a random Escherichia coli genomic library.

In order to identify new transmembrane helix packing motifs in naturally occurring proteins, we have selected transmembrane domains from a library of random Escherichia coli genomic DNA fragments and screened them for homomultimerization via their abilities to dimerize the bacteriophage lambda cI repressor DNA-binding domain. Sequences were isolated using a modified lambda cI headpiece dimerization assay system, which was shown previously to measure transmembrane helix-helix association in the E. coli inner membrane. Screening resulted in the identification of several novel sequences that appear to mediate helix-helix interactions. One sequence, representing the predicted sixth transmembrane domain (TM6) of the E. coli protein YjiO, was chosen for further analysis. Using site-directed mutagenesis and molecular dynamics, a small set of models for YjiO TM6 multimerization interface interactions were generated. This work demonstrates the utility of combining in vivo genetic tools with computational systems for understanding membrane protein structure and assembly.

Differential effects of surface and peat fire on soil constituents in a degraded wetland of the northern Florida Everglades.

The effects of surface (aboveground) and peat (belowground) fire on a number of soil constituents were examined within a hydrologically altered marsh in the northern Florida Everglades. Peat fire resulted in losses of total carbon (TC), total nitrogen (TN), and organic phosphorus (Po), while inorganic phosphorus (Pi) and total calcium (TCa) concentrations increased. In addition, peat fire led to a more pronounced vertical gradient in constituent concentrations between upper and lower soil layers. Surface fire also affected soil constituents, but impacts were small relative to peat fire. The effects of physical versus chemical processes during burning were assessed using ratios of constituent to TCa concentrations. This measure indicated that increases in the levels of total phosphorus (TP) in peat-burned areas were due primarily to the physical reduction of soil, while decreases in TN and TC were the result of volatilization. Increases in concentrations of Pi fractions arose from both chemically and physically mediated processes. In an ecological context, the observed soil transformations may encourage the growth of invasive plant species, such as southern narrow-leaved cattail (Typha domingensis Pers.), which exhibits high growth rates in response to increased P availability.

Lambda repressor N-terminal DNA-binding domain as an assay for protein transmembrane segment interactions in vivo.

To understand the determinants of membrane protein interactions, we have developed an in vivo genetic assay system for detecting homodimerization of transmembrane (TM) segments from integral membrane proteins. Our approach is to generate gene fusions between potentially dimerizing TM segments and a cytoplasmic DNA-binding protein that lacks its intrinsic dimerization domain. This genetic approach allows us to screen and distinguish among known dimerizing domains and weakly dimerizing mutants, as well as non-dimerizing TM segments. We replaced the bacteriophage lambda cI repressor C-terminal dimerization domain with the human erythrocyte glycophorin A transmembrane segment (GpA TM). GpA TM forms SDS-resistant homodimers in vitro. Expression of this membrane-associated fusion in Escherichia coli conferred the same degree of immunity to lambda cI phages as the wild-type, intact lambda repressor. Single amino acid substitutions that disrupt the GpA TM dimer interface were introduced into the lambda-GpA TM fusion proteins. These mutations dramatically reduced immunity of E. coli to lambda cI, such that the efficiency of plating these phages increased by greater than 10,000-fold over that conferred by the wild-type lambda-GpA TM fusion. Introduction of the putatively non-dimerizing first TM from E. coli MalF into the lambda-TM fusion vector resulted in no immunity to lambda cI phages. Fusion of the homodimeric, periplasmically localized, mature alkaline phosphatase domain to the C terminus of the lambda-TM fusion proteins containing weakly to non-dimerizing TM segments restored immunity to lambda cI phages. Results from this in vivo genetic assay system demonstrate that (1) dimerization of the lambda cI DNA-binding domain can be promoted by dimerizing TM segments, (2) strongly, weakly, and non-dimerizing TM segments can be distinguished on the basis of their ability to confer immunity to lambda cI phages, and (3) introduction of a dimerizing periplasmic domain can provide functionality to lambda-TM fusions containing weakly to non-dimerizing TM segments.

Enhancing transcription through the Escherichia coli hemolysin operon, hlyCABD: RfaH and upstream JUMPStart DNA sequences function together via a postinitiation mechanism.

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). The E. coli protein RfaH is required for wild-type hemolysin expression at the level of hlyCABD transcript elongation (J. A. Leeds and R. A. Welch, J. Bacteriol. 178:1850-1857, 1996). RfaH is also required for the transcription of wild-type levels of mRNA from promoter-distal genes in the rfaQ-K, traY-Z, and rplK-rpoC gene clusters, supporting the role for RfaH in transcriptional elongation. All or portions of a common 39-bp sequence termed JUMPStart are present in the untranslated regions of RfaH-enhanced operons. In this study, we tested the model that the JUMPStart sequence and RfaH are part of the same functional pathway. We examined the effect of JUMPStart deletion mutations within the untranslated leader of a chromosomally derived hlyCABD operon on hly RNA and HlyA protein levels in either wild-type or rfaH null mutant E. coli. We also provide in vivo physical evidence that is consistent with RNA polymerase pausing at the wild-type JUMPStart sequences.

RfaH enhances elongation of Escherichia coli hlyCABD mRNA.

Escherichia coli hlyCABD operons encode the polypeptide component (Hly A) 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.

Battling against host phagocytes: the wherefore of the RTX family of toxins?

The RTX family of bacterial exotoxins is a group of related cytolytic proteins produced by a wide variety of gram-negative human and animal pathogens. While diverse in their associated diseases and in their target cell specificities, there remain several themes common to RTX toxins, including genetic organization, structural and functional features, and effects on target cells. In this review, we summarize and discuss the genetics, regulation, epidemiology, structure/function relationships, and in vivo and in vitro activities of the best characterized RTX toxins, and speculate on their roles in pathogenesis and their use in immunotherapy.