The TolC Protein of Legionella pneumophila Plays a Major Role in Multi-Drug Resistance and the Early Steps of Host Invasion

Pneumonia associated with Iegionnaires's disease is initiated in humans after inhalation of contaminated aerosols. In the environment, Legionella pneumophila is thought to survive and multiply as an intracellular parasite within free-living amoeba. In the genome of L. pneumophila Lens, we identified a unique gene, tolC, encoding a protein that is highly homologous to the outer membrane protein TolC of Escherichia coli. Deletion of tolC by allelic exchange in L. pneumophila caused increased sensitivity to various drugs. The complementation of the tolC mutation in trans restored drug resistance, indicating that TolC is involved in multi-drug efflux machinery. In addition, deletion of tolC caused a significant attenuation of virulence towards both amoebae and macrophages. Thus, the TolC protein appears to play a crucial role in virulence which could be mediated by its involvement in efflux pump mechanisms. These findings will be helpful in unraveling the pathogenic mechanisms of L. pneumophila as well as in developing new therapeutic agents affecting the efflux of toxic compounds

An RND-Type Efflux System in Borrelia burgdorferi Is Involved in Virulence and Resistance to Antimicrobial Compounds

Borrelia burgdorferi is remarkable for its ability to thrive in widely different environments due to its ability to infect various organisms. In comparison to enteric Gram-negative bacteria, these spirochetes have only a few transmembrane proteins some of which are thought to play a role in solute and nutrient uptake and excretion of toxic substances. Here, we have identified an outer membrane protein, BesC, which is part of a putative export system comprising the components BesA, BesB and BesC. We show that BesC, a TolC homolog, forms channels in planar lipid bilayers and is involved in antibiotic resistance. A besC knockout was unable to establish infection in mice, signifying the importance of this outer membrane channel in the mammalian host. The biophysical properties of BesC could be explained by a model based on the channel-tunnel structure. We have also generated a structural model of the efflux apparatus showing the putative spatial orientation of BesC with respect to the AcrAB homologs BesAB. We believe that our findings will be helpful in unraveling the pathogenic mechanisms of borreliae as well as in developing novel therapeutic agents aiming to block the function of this secretion apparatus

C-signal: a cell surface-associated morphogen that induces and co-ordinates multicellular fruiting body morphogenesis and sporulation in Myxococcus xanthos

In Myxococcus xanthus, morphogenesis of multicellular fruiting bodies end sporulation are coordinated temporally and spatially, csgA mutants fail to synthesize the cell surface-associated C-signal and are unable to aggregate and sporulate, We report that csgA encodes two proteins, a 25 kDa species corresponding to full-length CsgA protein and a 17 kDa species similar in size to C-factor protein, which has been shown previously to have C-signal activity. By systematically varying the accumulation of the csgA proteins, we show that overproduction of the csgA proteins results in premature aggregation and sporulation, uncoupling of the two events and the formation of small fruiting bodies, whereas reduced synthesis of the csgA proteins causes delayed aggregation, reduced sporulation and the formation of large fruiting bodies, These results show that C-signal induces aggregation as well as sporulation, and that an ordered increase in the level of C-signalling during development is essential for the spatial co-ordination of these events. The results support a quantitative model, in which aggregation and sporulation are induced at distinct threshold levels of C-signalling. In this model, the two events are temporally co-ordinated by the regulated increase in C-signalling levels during development, The contact-dependent C-signal transmission mechanism allows the spatial co-ordination of aggregation and sporulation by coupling cell position and signalling levels

Coupling gene expression and multicellular morphogenesis during fruiting body formation in Myxococcus xanthus

A recurring theme in morphogenesis is the coupling of the expression of genes that drive morphogenesis and the morphogenetic process per se . This coupling ensures that gene expression and morphogenesis are carried out in synchrony. Morphogenesis of the spore-filled fruiting bodies in Myxococcus xanthus illustrates this coupling in the construction of a multicellular structure. Fruiting body formation involves two stages: aggregation of cells into mounds and the position-specific sporulation of cells that have accumulated inside mounds. Developmental gene expression propels these two processes. In addition, gene expression in individual cells is adjusted according to their spatial position. Progress in the understanding of the cell surface-associated C-signal is beginning to reveal the framework of an intercellular signalling system that allows the coupling of gene expression and multicellular morphogenesis. Accumulation of the C-signal is tightly regulated and involves transcriptional activation of the csgA gene and proteolysis of the full-length CsgA protein to produce the shorter cell surface-associated 17 kDa C-signal protein. The C-signal induces aggregation, sporulation and developmental gene expression at specific thresholds. The ordered increase in C-signalling levels, in combination with the specific thresholds, allows the C-signal to induce these three processes in the correct temporal order. The contact-dependent C-signal transmission mechanism, in turn, guarantees that C-signalling levels reflect the spatial position of individual cells relative to other cells and, thus, allows the cells to decode their spatial position during morphogenesis. By this mechanism, individual cells can tailor their gene expression profile to one that matches their spatial position. In this scheme, the molecular device that keeps gene expression in individual cells in register with morphogenesis is the C-signalling system, and the morphological structure, which is assessed, is the spatial position of individual cells relative to that of other cells

Crucial role of Asp408 in the proton translocation pathway of multidrug transporter AcrB: evidence from site-directed mutagenesis and carbodiimide labeling

The three-component AcrA/AcrB/TolC efflux system of Escherichia coli catalyzes the proton motive force-driven extrusion of a variety of cytotoxic compounds. The inner membrane pump component AcrB belongs to the resistance nodulation and cell division (RND) superfamily and is responsible for drug specificity and energy transduction of the entire tripartite efflux system. Systematic mutational analysis of titratable and polar membrane-located amino acids revealed four residues, D407, D408, K940, and, R971, to be of prime importance for AcrB function. Using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, D408 was shown to specifically react with dicyclohexylcarbodiimide (DCCD) in a pH-dependent manner. The apparent pK(a) of D408 of 7.4 would enable binding and release of protons under physiological conditions. In contrast to other secondary transporters, D408 was not protected from carbodiimide modification in the presence of drugs, which supports the notion of spatially separated transport pathways for drugs and protons. This study provides evidence for a substantial role of membrane-located carboxylates as a central element of the proton translocation pathway in AcrB and other members of the RND superfamily