An Arcanobacterium(Actinomyces) pyogenes Mutant Deficient in Production of the Pore-Forming Cytolysin Pyolysin Has Reduced Virulence

Abstract

Pyolysin (PLO), the hemolytic exotoxin expressed by Arcanobacterium (Actinomyces) pyogenes, is a member of the thiol-activated cytolysin family of bacterial toxins. Insertional inactivation of the plo gene results in loss of expression of PLO with a concomitant loss in hemolytic activity. The plo mutant, PLO-1, has an approximately 1.8-log 10 reduction in the 50% infectious dose compared to that for wild-type A. pyogenes in a mouse intraperitoneal infection model. Studies involving cochallenge of wild-type and PLO-1 bacteria resulted in recovery of similar numbers of both strains, suggesting that PLO production is required for survival in vivo. Recombinant, His-tagged PLO (His-PLO) is cytotoxic for mouse peritoneal macrophages and J774 cells in a dose-dependent manner. Protection against challenge with A. pyogenes could be afforded by vaccination with formalin-inactivated His-PLO, suggesting that PLO is a host-protective antigen, as well as a virulence determinant.

The gene encoding pyolysin, the pore-forming toxin ofArcanobacterium pyogenes, resides within a genomic islet flanked by essential genes

“…The former strategy attempted to disrupt the wild-type genes by allelic exchange, and the latter through a single crossover recombination event resulting in two defective copies of the target gene. Neither of these strategies resulted in viable recombinants with inactivated ftsY or ¡h genes, despite the fact that plo mutants of BBR1 could be isolated at a frequency of 2.5U10 2 mutants Wg 31 plasmid DNA using a previously constructed plo mutagenic plasmid, pJGS79 [5]. While these experiments do not directly con¢rm that ftsY and ¡h are essential in A. pyogenes, the number of failed attempts to construct stable mutants strongly suggests that they are essential under the conditions tested.…”

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“…However, we did not know if ALO was cytolytic to human leukocytes. Because of the lethal effects of other CDCs on human and mouse phagocytes, we hypothesized that ALO would act similarly, lysing human phagocytes and lymphocytes [ 19 - 22 ]. We assayed for the ability of purified rALO to lyse freshly isolated human neutrophils by incubating neutrophils under serum-free conditions with a range of rALO concentrations.…”

“…In vitro , under nutrient rich growth conditions, and in vivo B. anthracis expresses and secretes a protein that is a member of the CDC family of cytolysins, which we have named ALO [ 8 , 9 ]. CDCs are closely involved in the pathogenesis of several Gram-positive bacteria, acting both lytically to lyse cells as well as sub-lytically, modulating the immune response and basic immune cell function [ 14 - 19 , 23 - 26 , 29 , 31 ]. CDCs, including ALO, stimulate through TLR4.…”

“…CDCs are intimately involved in the pathogenesis of more than 20 Gram-positive species including Arcanobacterium pyogenes, Clostridium perfringens, Bacillus cereus, Listeria monocytogenes, Streptococcus pyogenes and Streptococcus pneumoniae [ 14 - 18 ]. At high concentrations, CDCs function as cytotoxins, lysing host immune cells; Perfringolysin O, PFO, is cytolytic to macrophages; Pneumolysin, PLY, lyses murine macrophages and bovine PMNs, and; SLO of S. pyrogenes lyses human PMNs [ 19 - 22 ]. Many of these pathogens secrete CDCs in sub-lytic amounts that function to modulate phagocytic cell function by inhibiting chemotaxis, the oxidative burst, or phagocytosis, and activate complement [ 12 , 15 , 23 - 27 ].…”

Experimental Infection of Archanobacterium Pyogenes in One Day Old Chicks

“…Five colonies from A.pyogeneswere inoculated in Brain heart infusion broth" BHIB", which incubate aerobically at 37°C for 24hours.A serial dilutionsin brain heart infusion broth was done, then 0.1 ml from each dilution tube is inoculated onthree nutrient agar platesand incubated aerobically at 37°C for 24 hours (12). Viable counts of bacteria were calculated and we selected the dose 4.2x10 8 CFU/ml for chicks and egg inoculation (13).…”