Screen for Leukotoxin Mutants in Aggregatibacter actinomycetemcomitans : Genes of the Phosphotransferase System Are Required for Leukotoxin Biosynthesis

Abstract

Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans is a pathogen that causes localized aggressive periodontitis and extraoral infections including infective endocarditis. Recently, we reported that A. actinomycetemcomitans is beta-hemolytic on certain growth media due to the production of leukotoxin (LtxA). Based on this observation and our ability to generate random transposon insertions in A. actinomycetemcomitans, we developed and carried out a rapid screen for LtxA mutants. Using PCR, we mapped several of the mutations to genes that are known or predicted to be required for LtxA production, including ltxA, ltxB, ltxD, and tdeA. In addition, we identified an insertion in a gene previously not recognized to be involved in LtxA biosynthesis, ptsH. ptsH encodes the protein HPr, a phosphocarrier protein that is part of the sugar phosphotransferase system. HPr results in the phosphorylation of other proteins and ultimately in the activation of adenylate cyclase and cyclic AMP (cAMP) production. The ptsH mutant showed only partial hemolysis on blood agar and did not produce LtxA. The phenotype was complemented by supplying wild-type ptsH in trans, and real-time PCR analysis showed that the ptsH mutant produced approximately 10-fold less ltxA mRNA than the wild-type strain. The levels of cAMP in the ptsH mutant were significantly lower than in the wild-type strain, and LtxA production could be restored by adding exogenous cAMP to the culture.

Correlations between Carbon Metabolism and Virulence in Bacteria

“…A ptsH mutant was reported to produce much less leukotoxin and to contain significantly lower cAMP levels. Adding exogenous cAMP could restore normal leukotoxin production [50]. Interestingly, in V. cholerae the PTS also plays a role in biofilm-associated growth.…”

Anti-leukemia activity of a bacterial toxin with natural specificity for LFA-1 on white blood cells

“…At the N-terminus are amphipathic helices that are believed to interact with the host cell membrane receptor and at the C-terminal half are nonapeptide glycine-rich repeats that are involved in calcium binding [13]. RTX toxins are secreted via an uncleaved C-terminal signal sequence by a type-I secretion mechanism [15] and we have recently characterized the components of this system in A. actinomycetemcomitans [16, 17]. Like HlyA and CyaA [18], LtxA is post-translationally modified at internal lysine residues with fatty acid moieties that are required for activity [19].…”

Aggregatibacter actinomycetemcomitans LtxC is required for leukotoxin activity and initial interaction between toxin and host cells

“…The gene for A. actinomycetemcomitans LtxA (ltxA) is a part of an operon containing a putative gene for LtxA modification (ltxC) (Kraig et al, 1990) and genes for secretion (ltxBD) (Isaza et al, 2008). A separate locus, tdeA, encoding a TolC-like protein, is also required for LtxA secretion (Crosby and Kachlany, 2007).…”