Expression of an antisense hla fragment in Staphylococcus aureus reduces alpha-toxin production in vitro and attenuates lethal activity in a murine model

Abstract

Isogeneic bacterial strains that differ only in the production of a single microbial factor have been invaluable in studying the pathogenesis of bacterial infections. The targeted, intentional inactivation of a gene encoding a potential virulence determinant generally requires homologous recombination to replace the gene with an inactivated allele. To determine whether the insertion and expression of a fragment of a bacterial gene in an antisense orientation could be used as a rapid alternative to allelic inactivation for producing paired isogeneic isolates, we inverted a 600-bp fragment of the Staphylococcus aureus gene encoding alpha-toxin, hla, behind its native promoter on an Escherichia coli-S. aureus shuttle vector. A transformant of an S. aureus strain carrying the antisense hla fragment produced antisense hla RNA and made 16-fold less alpha-toxin than either its parent or an isogeneic transformant containing vector DNA without hla. Also, intraperitoneal injection of 1.5 ؋ 10 9 CFU of the antisense hla-containing transformant was significantly less lethal in a murine model than that of the parent (1 of 10 versus 7 of 10 mice expired [P < 0.02]) or the transformant without hla (1 of 10 versus 7 of 7 mice expired [P < 0.001]). We conclude that the expression of a fragment of hla in an antisense orientation in S. aureus on a plasmid vector reduces alpha-toxin production and the lethal activity of the strain in a murine model. The antisense strategy for creating isogeneic strains of bacteria may facilitate molecular investigations into the pathogenesis of infection. It also may be useful in creating novel live-attenuated strains of bacteria for use as vaccine candidates.

Staphylococcus aureus α-toxin induces apoptosis in endothelial cells

“…aureus strains 8325-4, an K-toxin gene (hla)-positive laboratory strain derived from NCTC 8325, and its hlanegative isogenic derivative DU1090 constructed by allelic replacement were obtained from T. Foster (Dublin) [12]. S. aureus strain DK2076(pVK151) in which the chromosomal hla expression is attenuated by antisense vector construction and its parent DK2076 were also used [13]. The strains were grown overnight (18 h) in BHI at 37³C with vigorous shaking.…”

“…To con¢rm the e¡ect of K-toxin expression upon the induction of apoptosis in endothelial cells in vitro, S. aureus DK2076(pVK151) in which the production of K-toxin is downregulated by cloning of an antisense fragment of hla [13] was compared to its parent DK2076, a clinical S. aureus isolate that is virulent in laboratory animals. Antisense methods have been successfully used to downregulate prokaryotic gene expression including that of hla in S. aureus [13,16]. The antisense-constructed strain DK2076(pVK151) makes 16-fold less K-toxin than its parent DK2076 [13].…”

“…Antisense methods have been successfully used to downregulate prokaryotic gene expression including that of hla in S. aureus [13,16]. The antisense-constructed strain DK2076(pVK151) makes 16-fold less K-toxin than its parent DK2076 [13]. Con£uent endothelial cell monolayers were incubated for 1 h with a bacterial inoculum to achieve a MOI of 10:1, washed three times with M199, and then incubated in the presence of lysostaphin until harvested for endothelial cell genomic DNA extraction.…”

Zoocin A facilitates the entry of antisense constructs into Streptococcus mutans

“…Antisense asODNs have been used with some success to downregulate gene expression in a variety of bacteria, such as S. mutans (Baev et al, 1999;Wang & Kuramitsu, 2003), S. aureus (Kernodle et al, 1997;Ji et al, 2002), and mycobacteria (Parish & Stoker, 1997;Wilson et al, 1998). It has, however, proven difficult to overcome diffusion limitations imposed by the outer lipopolysaccaride membrane of Gram-negative bacteria and the thick peptidoglycan layer of Gram-positive bacteria.…”

Characterisation of anti‐alpha toxin antibody levels and colonisation status after administration of an investigational human monoclonal antibody, MEDI4893, against Staphylococcus aureus alpha toxin

“…[6][7][8][9][10][11] Staphylococcus aureus strains deficient in AT expression are less virulent in animal models of dermonecrosis, pneumonia, sepsis, endocarditis and mastitis. [11][12][13][14][15][16][17] Targeted neutralisation of AT could therefore prevent or limit S. aureus disease. This hypothesis is supported by studies that demonstrated a reduction in S. aureus disease severity in murine infection models after active or passive immunisation directed against AT.…”