Isolation and molecular characterization of spontaneously occurring cytolysin-negative mutants of Actinobacillus pleuropneumoniae serotype 7

Abstract

Actinobacillus pleuropneumoniae serotype 7 strains are shown to spontaneously lose cytolytic activity, with a frequency of approximately 10-4. The phenotypic change is associated with the loss of approximately 8.5 kbp of chromosomal DNA. A genomic fragment encoding the cytolysin and its flanking sequences was cloned and characterized. Also, the corresponding truncated fragment was cloned from a spontaneous mutant. Comparison of the two clones allowed the definition of the excision site. The ends of the excised fragment are composed of 1,201 bp long direct identical repeats, possibly facilitating the genotypic change by homologous recombination. In accordance with this hypothesis, one repeat is conserved in the spontaneous mutant. Each repeat contains one open reading frame preceded by a Shine-Dalgarno consensus sequence, and the ends of each repeat contain 26-bp complementary sequences with four mismatches.Rearrangement of bacterial DNA as a means to increase the fitness of survival of the respective bacterial population is a relatively common phenomenon. Examples are the genetic switch resulting in the expression of an antigenically distinct flagella protein (H-antigen) in Salmonella species (25), the on/off switch of type 1 fimbriae in Escherichia coli (1), and the chromosomal rearrangements leading to the expression of antigenically distinct fimbriae in Moraxella bovis (18) and Neisseria gonorrhoeae (23). More recently, the site-specific integration of the virulence plasmid of enteroinvasive E. coli and Shigellaflexneri into the chromosome has been described; this results in a noninvasive and thus avirulent phenotype (36). Also, high-frequency excision of mobile genetic elements under conditions of nutritional stress has been described; this restored the relevant catabolic genes to normal function (8,24). Insertion and excision events are also encountered in combination with horizontal transfer of virulence factors. Thus, the heat-stable toxins of enterotoxigenic E. coli (STI and STII) can be carried by transposable elements, and the inverted repeats in these elements are composed of insertion sequences IS] and IS2, respectively (11,15,27,28).The cytolysins or RTX toxins form a large family of calcium-dependent, pore-forming toxins which can be found in different genera of the families Enterobacteriaceae and Pasteurellaceae (34). Genetic investigation of these toxins has shown that the toxin-encoding gene (hlyA in E. coli) is commonly clustered with at least three other genes encoding an activator (HlyC) and proteins involved in membrane transport (HlyB and HlyD [5,35]). A comparison of the nucleotide and amino acid sequences of RTX toxins from different species shows a high degree of similarity, thus suggesting the occurrence of horizontal transfer rather than parallel evolutionary evolvement (2,5,6,16,34).In Actinobacillus pleuropneumoniae at least three immunologically and functionally distinct cytolysins of + 110 x 103, + 105 x 103, and + 103 x 103 Da (+110K, 105K, and * Corresponding author. t VIDO Journa...

Actinobacillus pleuropneumoniaeserotype 7 siderophore receptorFhuAis not required for virulence

“…Escherichia coli strains were cultured in Luria^Bertani (LB) medium supplemented with the appropriate antibiotics (ampicillin, 100 Wg ml 31 ; kanamycin, 50 Wg ml 31 ); for cultivation of E. coli L2155 (vdapA), diaminopimelic acid (1 mM; Sigma Chemical Co., Deisenhofen, Germany) was added. A. pleuropneumoniae strains were cultured in PPLO medium (Difco, Augsburg, Germany) supplemented with NAD (10 Wg ml 31 ; E. Merck, Darmstadt, Germany), L-glutamine (100 Wg ml 31 ; Serva, Heidelberg, Germany), L-cysteine hydrochloride (260 Wg ml 31 ; Sigma), L-cystine dihydrochloride (10 Wg ml 31 ; Sigma), dextrose (1 mg ml 31 ), and Tween 0 80 (0.1%). For the selection of A. pleuropneumoniae transconjugants, chloramphenicol (5 Wg ml 31 ) was added.…”

Induction of protective immune responses against the challenge ofActinobacillus pleuropneumoniaeby the oral administration of transgenic tobacco plant expressing ApxIIA toxin from the bacteria

“…Although multiple factors are believed to be involved in the virulence of A. pleuropneumoniae infection and the exact mechanism of infection is not yet completely understood, previous studies indicated that Apx toxins correlate strongly with the virulence of the bacterium (Tascon et al, 1994;Frey, 1995;Reimer et al, 1995). The Apx toxins are thought to be of particular importance among the virulence factors because the toxins can induce protective immune responses against the bacterial infection, as demonstrated previously using several mutants (Anderson et al, 1991;Tascon et al, 1994;Reimer et al, 1995;Prideaux et al, 1999;Fuller et al, 2000).…”

Vaccination and Protection of Pigs against Pleuropneumonia with a Vaccine Strain of Actinobacillus pleuropneumoniaeProduced by Site-Specific Mutagenesis of the ApxII Operon

“…Previously we had administered the toxin-deficient strain HS93 Tox Ϫ to pigs at doses similar to that of the challenge used in this experiment and autopsied the pigs at day 5 but observed no lesions. Apxdeficient mutants of APP produced by either chemical or transposon mutagenesis have previously been shown to have a reduced ability to induce lung lesions (1,15,17,29,30,33,34). The six unvaccinated pigs challenged with HS25 showed numerous lung lesions that were visible on autopsy, indicating that the level of challenge used was sufficient to induce lesions in unprotected animals.…”

“…These secreted toxins, or Apx toxins, are members of the RTX toxin family (11)(12)(13). The role of Apx toxins in A. pleuropneumoniae virulence was first demonstrated with spontaneous and chemically induced nonhemolytic mutants which were found to be completely or partially avirulent; this role was later confirmed by using transposon mutagenesis (1,15,17,29,30,33,34). At least three different Apx toxins are produced by A. pleuropneumoniae, designated ApxI, ApxII, and ApxIII.…”