Synthesis and antibacterial activity of novel modified 5-O-desosamine ketolides.

A series of novel modified 5-O-desosamine-ketolides were synthesized. The 5-O-desosamine fragment was removed from ketolide by an efficient and mild manipulation. 4-O-substituted desosamine was introduced into the ketolide aglycon and various coupling methods were essayed for the glycosylation. Three novel ketolides were tested for in vitro antibacterial activity against a panel of susceptible and resistant pathogens. Compound 26 showed potent activity against all the methicillin-sensitivity and resistant pathogens.

Acyl-sulfamates target the essential glycerol-phosphate acyltransferase (PlsY) in Gram-positive bacteria.

PlsY is the essential first step in membrane phospholipid synthesis of Gram-positive pathogens. PlsY catalyzes the transfer of the fatty acid from acyl-phosphate to the 1-position of glycerol-3-phosphate to form the first intermediate in membrane biogenesis. A series of non-metabolizable, acyl-sulfamate analogs of the acyl-phosphate PlsY substrate were prepared and evaluated as inhibitors of Staphylococcus aureus PlsY and for their Gram-positive antibacterial activities. From this series phenyl (8-phenyloctanoyl) sulfamate had the best overall profile, selectively inhibiting S. aureus phospholipid biosynthesis and causing the accumulation of both long-chain fatty acids and acyl-acyl carrier protein intermediates demonstrating that PlsY was the primary cellular target. Bacillus anthracis was unique in being more potently inhibited by long chain acyl-sulfamates than other bacterial species. However, it is shown that Bacillus anthracis PlsY is not more sensitive to the acyl-sulfamates than S. aureus PlsY. Metabolic profiling showed that B. anthracis growth inhibition by the acyl-sulfamates was not specific for lipid synthesis illustrating that the amphipathic acyl-sulfamates can also have off-target effects in Gram-positive bacteria. Nonetheless, this study further advances PlsY as a druggable target for the development of novel antibacterial therapeutics, through the discovery and validation of the probe compound phenyl (8-phenyloctanoyl) sulfamate as a S. aureus PlsY inhibitor.

Phage-associated mutator phenotype in group A streptococcus.

Defects in DNA mismatch repair (MMR) occur frequently in natural populations of pathogenic and commensal bacteria, resulting in a mutator phenotype. We identified a unique genetic element in Streptococcus pyogenes strain SF370 that controls MMR via a dynamic process of prophage excision and reintegration in response to growth. In S. pyogenes, mutS and mutL are organized on a polycistronic mRNA under control of a common promoter. Prophage SF370.4 is integrated between the two genes, blocking expression of the downstream gene (mutL) and resulting in a mutator phenotype. However, in rapidly growing cells the prophage excises and replicates as an episome, allowing mutL to be expressed. Excision of prophage SF370.4 and expression of MutL mRNA occur simultaneously during early logarithmic growth when cell densities are low; this brief window of MutL gene expression ends as the cell density increases. However, detectable amounts of MutL protein remain in the cell until the onset of stationary phase. Thus, MMR in S. pyogenes SF370 is functional in exponentially growing cells but defective when resources are limiting. The presence of a prophage integrated into the 5' end of mutL correlates with a mutator phenotype (10(-7) to 10(-8) mutation/generation, an approximately a 100-fold increase in the rate of spontaneous mutation compared with prophage-free strains [10(-9) to 10(-10) mutation/generation]). Such genetic elements may be common in S. pyogenes since 6 of 13 completed genomes have related prophages, and a survey of 100 strains found that about 20% of them are positive for phages occupying the SF370.4 attP site. The dynamic control of a major DNA repair system by a bacteriophage is a novel method for achieving the mutator phenotype and may allow the organism to respond rapidly to a changing environment while minimizing the risks associated with long-term hypermutability.

Prophage induction and expression of prophage-encoded virulence factors in group A Streptococcus serotype M3 strain MGAS315.

The genome of the highly virulent group A Streptococcus (GAS) serotype M3 strain MGAS315 has six prophages that encode six proven or putative virulence factors. We examined prophage induction and expression of prophage-encoded virulence factors by this strain under in vitro conditions inferred to approximate in vivo conditions. Coculture of strain MGAS315 with Detroit 562 (D562) human epithelial pharyngeal cells induced the prophage encoding streptococcal pyrogenic exotoxin K (SpeK) and extracellular phospholipase A(2) (Sla) and the prophage encoding streptodornase (Sdn). Increased gene copy numbers after induction correlated with increased speK, sla, and sdn transcript levels. Although speK and sla are located contiguously in prophage Phi315.4, these genes were transcribed independently. Whereas production of immunoreactive SpeK was either absent or minimal during coculture of GAS with D562 cells, production of immunoreactive Sla increased substantially. In contrast, despite a lack of induction of the prophage encoding speA during coculture of GAS with D562 cells, the speA transcript level and production of immunoreactive streptococcal pyrogenic exotoxin A (SpeA) increased. Exposure of strain MGAS315 to hydrogen peroxide, an oxidative stressor, induced the prophage encoding mitogenic factor 4 (MF4), and there was a concomitant increase in the mf4 transcript. All prophages of strain MGAS315 that encode virulence factors were induced during culture with mitomycin C, a DNA-damaging agent. However, the virulence factor gene transcript levels and production of the encoded proteins decreased after mitomycin C treatment. Taken together, the results indicate that a complex relationship exists among environmental culture conditions, prophage induction, and production of prophage-encoded virulence factors.

A fluoroquinolone induces a novel mitogen-encoding bacteriophage in Streptococcus canis.

This study investigated whether the recently recognized emergence of canine streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) might be partly attributed to the use of fluoroquinolones to treat Streptococcus canis infections in dogs. Both mitomycin and the fluoroquinolone enrofloxacin caused bacteriophage-induced lysis of S. canis strain 34, an isolate from a case of canine STSS and NF. Fluoroquinolone-evoked, bacteriophage-induced lysis occurred over a range of concentrations similar to those that would occur after treatment of dogs with these agents. To search for a possible bacteriophage-encoded streptococcal superantigen gene(s), a library of the 36.5 (+/-1.1)-kb bacteriophage, designated phisc1, was made by ligating 3- to 7-kb Tsp5091-digested phisc1 fragments into an EcoRI-digested lambdaZapII vector. Recombinants were screened for mitogenic activity by using canine peripheral blood lymphocytes. Of 800 recombinants screened, 11 recombinants with mitogenic effects were identified, and their inserts were sequenced. The highest homology of 11.6 kb of sequenced phisc1 DNA was to the completely sequenced Streptococcus pneumoniae bacteriophage MM1. Seven of the 11 phisc1 sequenced inserts contained a 552-bp open reading frame, scm, with 27% amino acid similarity to pokeweed (Phytolacca americana) mitogen. PCR showed this gene to be present in 22 of 23 S. canis isolates tested. Quantitative reverse transcription-PCR showed that bacteriophage induction was associated with a 58-fold enhancement of expression of this gene relative to that in a noninduced culture of a similar age. The presence of this gene on a fluoroquinolone-induced bacteriophage may explain the association observed between fluoroquinolone use in dogs and the development of canine STTS and NF.