Synthesis and Evaluation of Thiazolidine Amide and N-Thiazolyl Amide Fluoroquinolone Derivatives.

In an effort to develop new fluoroquinolones, we synthesized eight compounds and tested them against a panel of bacteria. The design of these compounds was guided by the introduction of the isothiazoloquinolone motif. The three most active compounds in this series, 8-10, demonstrated good antibacterial activity against methicillin-sensitive Staphylococcus aureus and healthcare-acquired methicillin-resistant Staphylococcus aureus (MIC 0.62-6.3 µg/mL). Further, when these three active compounds were tested for their inhibitory effects on bacterial enzymes, compound 9 was the most effective agent exhibiting IC50 values of 33.9 and 116.5 µM in the S. aureus deoxyribonucleic acid (DNA) gyrase supercoiling and topoisomerase IV decatenation assays, respectively.

A putative transmembrane leucine zipper of agrobacterium VirB10 is essential for t-pilus biogenesis but not type IV secretion.

The Agrobacterium tumefaciens VirB/VirD4 type IV secretion system is composed of a translocation channel and an extracellular T pilus. Bitopic VirB10, the VirB7 lipoprotein, and VirB9 interact to form a cell envelope-spanning structural scaffold termed the "core complex" that is required for the assembly of both structures. The related pKM101-encoded core complex is composed of 14 copies each of these VirB homologs, and the transmembrane (TM) α helices of VirB10-like TraF form a 55-Å-diameter ring at the inner membrane. Here, we report that the VirB10 TM helix possesses two types of putative dimerization motifs, a GxxxA (GA4) motif and two leucine (Leu1, Leu2) zippers. Mutations in the Leu1 motif disrupted T-pilus biogenesis, but these or other mutations in the GA4 or Leu2 motif did not abolish substrate transfer. Replacement of the VirB10 TM domain with a nondimerizing poly-Leu/Ala TM domain sequence also blocked pilus production but not substrate transfer or formation of immunoprecipitable complexes with the core subunits VirB7 and VirB9 and the substrate receptor VirD4. The VirB10 TM helix formed weak homodimers in Escherichia coli, as determined with the TOXCAT assay, whereas replacement of the VirB10 TM helix with the strongly dimerizing TM helix from glycophorin A blocked T-pilus biogenesis in A. tumefaciens. Our findings support a model in which VirB10's TM helix contributes to the assembly or activity of the translocation channel as a weakly self-interacting membrane anchor but establishes a heteromeric TM-TM helix interaction via its Leu1 motif that is critical for T-pilus biogenesis.

Agrobacterium VirB10 domain requirements for type IV secretion and T pilus biogenesis.

Agrobacterium tumefaciens VirB10 couples inner membrane (IM) ATP energy consumption to substrate transfer through the VirB/D4 type IV secretion (T4S) channel and also mediates biogenesis of the virB-encoded T pilus. Here, we determined the functional importance of VirB10 domains denoted as the: (i) N-terminal cytoplasmic region, (ii) transmembrane (TM) alpha-helix, (iii) proline-rich region (PRR) and (iv) C-terminal beta-barrel domain. Mutations conferring a transfer- and pilus-minus (Tra(-), Pil(-)) phenotype included PRR deletion and beta-barrel substitution mutations that prevented VirB10 interaction with the outer membrane (OM) VirB7-VirB9 channel complex. Mutations permissive for substrate transfer but blocking pilus production (Tra(+), Pil(-)) included a cytoplasmic domain deletion and TM domain insertion mutations. Another class of Tra(+) mutations also selectively disrupted pilus biogenesis but caused release of pilin monomers to the milieu; these mutations included deletions of alpha-helical projections extending from the beta-barrel domain. Our findings, together with results of Cys accessibility studies, indicate that VirB10 stably integrates into the IM, extends via its PRR across the periplasm, and interacts via its beta-barrel domain with the VirB7-VirB9 channel complex. The data further support a model that distinct domains of VirB10 regulate formation of the secretion channel or the T pilus.