Infectivity of a glucan synthesis-defective mutant of Streptococcus gordonii (Challis) in a rat endocarditis model

Streptococcus gordonii, a member of the human indigenous oral microflora, colonizes smooth tooth surfaces and contributes to dental plaque formation. Although it is not recognized as being a cariogenic pathogen, it may cause endocarditis following invasion of the bloodstream. Using allelic exchange mutagenesis, we have constructed a mutant of S. gordonii (Challis) which is defective in its single functional glucosyltransferase gene and, hence, is unable to synthesize glucan exopolymers from sucrose. When examined in a rat endocarditis model, the sucrose-grown mutant did not differ significantly from S. gordonii wild-type, suggesting that glucan polymers did not contribute to infectivity. This result was in striking contrast to that previously observed with a polymer-defective S. mutans mutant.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30573/1/0000208.pd

Regulation of glucosyltransferase in Streptococcus gordonii.

Streptococcus gordonii is found in dental plaque in the oral cavity, and it is also associated with infective endocarditis. It exhibits a hard colony phenotype (Spp\sp{+}) on media containing sucrose; however, soft colony variants (Spp\sp{-}) appeared at a frequency of 10\sp{-4} to 10\sp{-3}. Selected Spp\sp{-} strains were able to revert to Spp\sp{+} at a similar frequency. A correlation between change in phenotype and extracellular glucosyltransferase (GTF) activity was observed. In order to determine a possible molecular basis for the phase transition, genetic analysis of the GTF locus was conducted. A structural gtf gene was cloned, and the N-terminal portion (about 1/3 of the gene) was sequenced. A region of about 1 kb displayed $>$50% amino acid identity with gtf genes identified in Streptococcus mutans and Streptococcus downei. Insertional mutagenesis of the gene resulted in a strain with an Spp\sp{-} phenotype and no detectable GTF activity. The gene was designated gtfG. A regulatory gene, rgg, was identified 66-bp upstream of gtfG. When present in multiple copies on a plasmid chimera, rgg functionally complemented Spp\sp{-} variants, and in an Spp\sp{+} host it increased GTF expression well above (7 to 12 fold) wild type levels. Disruption of the chromosomal rgg gene in an Spp\sp{+} strain resulted in an Spp\sp{-} phenotype exhibiting only 3% of wild-type GTF activity. Northern analysis showed that rgg positively regulates transcription of gtfG. Nucleotide sequencing revealed two inverted repeats located before rgg and intercistronically between rgg and gtfG, both of which may play a role in the regulation of gtfG. Thus, rgg and gtfG are necessary for GTF expression and the Spp\sp{+} phenotype. Comparison of chromosomal DNA from S. gordonii Spp\sp{+} and Spp\sp{-} phase variants by Southern blot analysis using a variety of different restriction enzymes resolved no large DNA rearrangements at the rgg/gtfG locus. Northern analysis identified a decrease in the rgg-positively-regulated gtfG message in only a subset of Spp\sp{-} variants. Spp variants therefore may be generated by more than one type of genetic event, one of which involves altered levels of a positively-regulated gtfG RNA transcript.Ph.D.Microbiology and ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103320/1/9308461.pdfDescription of 9308461.pdf : Restricted to UM users only

Lesions in Teichoic Acid Biosynthesis in Staphylococcus aureus Lead to a Lethal Gain of Function in the Otherwise Dispensable Pathway

An extensive study of teichoic acid biosynthesis in the model organism Bacillus subtilis has established teichoic acid polymers as essential components of the gram-positive cell wall. However, similar studies pertaining to therapeutically relevant organisms, such as Staphylococcus aureus, are scarce. In this study we have carried out a meticulous examination of the dispensability of teichoic acid biosynthetic enzymes in S. aureus. By use of an allelic replacement methodology, we examined all facets of teichoic acid assembly, including intracellular polymer production and export. Using this approach we confirmed that the first-acting enzyme (TarO) was dispensable for growth, in contrast to dispensability studies in B. subtilis. Upon further characterization, we demonstrated that later-acting gene products (TarB, TarD, TarF, TarIJ, and TarH) responsible for polymer formation and export were essential for viability. We resolved this paradox by demonstrating that all of the apparently indispensable genes became dispensable in a tarO null genetic background. This work suggests a lethal gain-of-function mechanism where lesions beyond the initial step in wall teichoic acid biosynthesis render S. aureus nonviable. This discovery poses questions regarding the conventional understanding of essential gene sets, garnered through single-gene knockout experiments in bacteria and higher organisms, and points to a novel drug development strategy targeting late steps in teichoic acid synthesis for the infectious pathogen S. aureus

In Vitro and In Vivo Activities of PD 0305970 and PD 0326448, New Bacterial Gyrase/Topoisomerase Inhibitors with Potent Antibacterial Activities versus Multidrug-Resistant Gram-Positive and Fastidious Organism Groups

PD 0305970 and PD 0326448 are new bacterial gyrase and topoisomerase inhibitors (quinazoline-2,4-diones) that possess outstanding in vitro and in vivo activities against a wide spectrum of bacterial species including quinolone- and multidrug-resistant gram-positive and fastidious organism groups. The respective MICs (μg/ml) for PD 0305970 capable of inhibiting ≥90% of bacterial strains tested ranged from 0.125 to 0.5 versus staphylococci, 0.03 to 0.06 versus streptococci, 0.25 to 2 versus enterococci, and 0.25 to 0.5 versus Moraxella catarrhalis, Haemophilus influenzae, Listeria monocytogenes, Legionella pneumophila, and Neisseria spp. PD 0326448 MIC(90)s were generally twofold higher versus these same organism groups. Comparative quinolone MIC(90) values were 4- to 512-fold higher than those of PD 0305970. In testing for frequency of resistance, PD 0305970 and levofloxacin showed low levels of development of spontaneous resistant mutants versus both Staphylococcus aureus and Streptococcus pneumoniae. Unlike quinolones, which target primarily gyrA and parC, analysis of resistant mutants in S. pneumoniae indicates that the likely targets of PD 0305970 are gyrB and parE. PD 0305970 demonstrated rapid bactericidal activity by in vitro time-kill testing versus streptococci. This bactericidal activity carried over to in vivo testing, where PD 0305970 and PD 0326448 displayed outstanding Streptococcus pyogenes 50% protective doses (PD(50)s) (oral dosing) of 0.7 and 3.6 mg/kg, respectively (ciprofloxacin and levofloxacin PD(50)s were >100 and 17.7 mg/kg, respectively). PD 0305970 was also potent in a pneumococcal pneumonia mouse infection model (PD(50) = 3.2 mg/kg) and was 22-fold more potent than levofloxacin

Genetic Footprinting in Bacteria

In vivo genetic footprinting was developed in the yeast Saccharomyces cerevisiae to simultaneously assess the importance of thousands of genes for the fitness of the cell under any growth condition. We have developed in vivo genetic footprinting for Escherichia coli, a model bacterium and pathogen. We further demonstrate the utility of this technology for rapidly discovering genes that affect the fitness of E. coli under a variety of growth conditions. The definitive features of this system include a conditionally regulated Tn10 transposase with relaxed sequence specificity and a conditionally regulated replicon for the vector containing the transposase and mini-Tn10 transposon with an outwardly oriented promoter. This system results in a high frequency of randomly distributed transposon insertions, eliminating the need for the selection of a population containing transposon insertions, stringent suppression of transposon mutagenesis, and few polar effects. Successful footprints have been achieved for most genes longer than 400 bp, including genes located in operons. In addition, the ability of recombinant proteins to complement mutagenized hosts has been evaluated by genetic footprinting using a bacteriophage λ transposon delivery system

A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore

As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious Gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity