Increased sensitivity of Aggregatibacter actinomycetemcomitans to human serum is mediated by induction of a bacteriophage

Abstract

Aggregatibacter actinomycetemcomitans, a Gram-negative oral pathobiont causing aggressive periodontitis and systemic infections, demonstrates serum resistance. We have identified a dsDNA-tailed bacteriophage, S1249, which was found to convert from this microorganism inducible by human serum into a lytic state to kill the bacterium. This phage demonstrated active transcripts when exposed to human serum: 20% of genes were upregulated more than 10-fold, and 45% of them were upregulated 5-10-fold when the bacterium was grown in the presence of human serum compared to without the presence of human serum. Transcriptional activation when grown in equine serum was less pronounced. This phage demonstrated a tail with inner rigid tubes and an outer contractile sheath, features of Myoviridae spp. Further characterization revealed that the lysogenized integration of the phage in the chromosome of A. actinomycetemcomitans occurred between the genes encoding cold-shock DNAbinding domain-containing protein (csp) and glutamyl-tRNA synthetase (gltX). Both phage DNA integrated lysogeny and nonintegrated pseudolysogeny were identified in the infected bacterium. A newly generated, lysogenized strain using this phage displayed similar attributes, including 63% growth inhibition compared to its isogenic phage-free strain when in the presence of human serum. Our data suggest that bacteriophage S1249 can be induced in the presence of human serum and enters the lytic cycle, which reduces the viability of infected bacteria in vivo.

Human Serum Mediated Bacteriophage Life Cycle Switch in Aggregatibacter actinomycetemcomitans Is Linked to Pyruvate Dehydrogenase Complex

“…A. actinomycetemcomitans was routinely recovered from the freezer using TSBYE agar, containing 3% trypticase soy broth, 0.6% yeast extract, and 1.5% agar (Becton Dickinson and Company, Franklin Lakes, NJ, USA) and incubated statically in a 37 °C incubator with 5% humidified carbon dioxide atmosphere for each experiment. The phage-naïve A. actinomycetemcomitans strain IDH84 of serotype c [ 14 , 35 ] and its derived lysogenized strain IDH84/S1249 were used for this investigation. The strain IDH84/S1249 was generated by infecting strain IDH84 with the isolated bacteriophage S1249, which demonstrated phage DNA integration into the bacterial chromosome [ 35 ].…”

“…The phage-naïve A. actinomycetemcomitans strain IDH84 of serotype c [ 14 , 35 ] and its derived lysogenized strain IDH84/S1249 were used for this investigation. The strain IDH84/S1249 was generated by infecting strain IDH84 with the isolated bacteriophage S1249, which demonstrated phage DNA integration into the bacterial chromosome [ 35 ].…”

“…Aggregatibacter phage S1249 was isolated from its originally identified clinical lysogenic strain D11S-1 [ 32 , 35 , 42 ], using a modified protocol described by Tang-Siegel et al [ 35 ]. Briefly, the overnight culture of 10 mL bacterial lysogen grown at 37 °C with 5% humidified CO 2 atmosphere using TSBYE was diluted in 100 mL, grown for two doubling-time (6 h), and exposed to 0.5 μg/mL mitomycin C from Streptomyces caespitosus (M0503; Sigma-Aldrich, St Louis, MO, USA) for 30 min.…”

“…We, however, have identified a pseudolysogenic phage S1249, which increases the sensitivity of A. actinomycetemcomitans to human serum [ 32 , 33 , 34 , 35 ]. Over 20% of phage genes were up-regulated at least 10-fold with a concomitant increase in the kinetics of phage egress, after the exposure of its lysogen to human serum [ 33 , 35 ], including a cluster of phage regulatory factors [ 35 ]. Interestingly, sera from other animals, including equines, do not stimulate the same response [ 32 , 33 , 35 ].…”

“…Over 20% of phage genes were up-regulated at least 10-fold with a concomitant increase in the kinetics of phage egress, after the exposure of its lysogen to human serum [ 33 , 35 ], including a cluster of phage regulatory factors [ 35 ]. Interestingly, sera from other animals, including equines, do not stimulate the same response [ 32 , 33 , 35 ]. Bacteriophages have been emerging as alternative therapeutics to treat certain bacterial infection to combat the rising antibiotic resistance [ 36 ], as well as a potential delivery platform for future vaccine developments [ 37 ].…”