The in vitro interaction of Streptococcus pyogenes with human pharyngeal cells induces a phage-encoded extracellular DNase - PubMed (original) (raw)

The in vitro interaction of Streptococcus pyogenes with human pharyngeal cells induces a phage-encoded extracellular DNase

Thomas B Broudy et al. Infect Immun. 2002 Jun.

Abstract

The role lysogenic bacteriophage play in the pathogenesis of the host bacterium is poorly understood. In a previous study, we found that streptococcal coculture with human pharyngeal cells resulted in the induction of lysogenic bacteriophage as well as the phage-associated streptococcal pyrogenic exotoxin C (SpeC). In this study, we have determined that in addition to SpeC induction, a number of other streptococcal proteins are also released by the bacteria during coculture with pharyngeal cells. Among these, we identified and characterized a novel 27-kDa secreted protein. Sequence analysis of this novel protein demonstrated it to be encoded by the same lysogenic bacteriophage which harbors speC. Protein sequence analysis revealed varied homologies with several streptococcal DNases. Further biochemical characterization of the recombinantly expressed protein verified it to be a divalent cation-dependent streptococcal phage-encoded DNase (Spd1). Although functionally distinct, SpeC and Spd1 are associated by a number of parameters, including genetic proximity and transcriptional regulation. Finally, we speculate on the induction of phage-encoded DNase (Spd1) enhancing the fitness of both bacteria and phage.

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Figures

FIG. 1.

FIG. 1.

[35S]Met-labeled proteins in the culture media of the non-cycloheximide-treated pharyngeal-cell control [Detroit only (− cyclo)] and the cycloheximide-treated samples, including the pharyngeal-cell control [Detroit only (+ cyclo)], pharyngeal-cell-S. pyogenes coculture (Detroit/D471), and S. pyogenes control (D471 only).

FIG. 2.

FIG. 2.

Schematic alignment of spd1 and speC sequences from the integrated prophage in the M1 genome and the region of the M6 strain D471 which was sequenced. attL and attR, left and right attachment sites, respectively, of the integrated prophage.

FIG. 3.

FIG. 3.

Southern blot of _Hin_dIII-digested genomic DNAs from 19 strains of S. pyogenes probed with either spd1 or speC. The M serotype is designated in parentheses next to the strain number, and the strains are grouped by their surface-exposed antigenic properties as either class I or class II. The DNA ladder is shown to the left, with arrows indicating 1-kb increments.

FIG. 4.

FIG. 4.

RNAs extracted from Detroit 562 cells, D471 cocultured with Detroit 562 (Cocultured D471), and D471 alone were blotted and probed for either spd1 or speC.

FIG. 5.

FIG. 5.

Culture supernatants from S. gordonii (GP251) parent strain and supernatants from recombinant S. gordonii (secreting either NucA or Spd1) were collected. Each supernatant was incubated with pBlueScript DNA in Tris-Ca2+-Mg2+ buffer, either with (+) or without (−) EDTA present. The completed reaction was analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining.

FIG. 6.

FIG. 6.

(A) Amino acid sequence alignment of the Spd1 nuclease with the known nuclease MF/DNase B and three other putative DNases, including two from S. pyogenes (MF2 and MF3) and one from L. lactis (YbfB). The conserved histidine is indicated with an arrowhead. The dots represent spaces in the sequence alignment. Solid boxes represent amino acid identity, and shaded boxes represent amino acid similarity based on Clustal W sequence analysis. (B) Amino acid sequence identities between MF/DNase B or Spd1 and the Spd1-related putative DNases.

FIG. 6.

FIG. 6.

(A) Amino acid sequence alignment of the Spd1 nuclease with the known nuclease MF/DNase B and three other putative DNases, including two from S. pyogenes (MF2 and MF3) and one from L. lactis (YbfB). The conserved histidine is indicated with an arrowhead. The dots represent spaces in the sequence alignment. Solid boxes represent amino acid identity, and shaded boxes represent amino acid similarity based on Clustal W sequence analysis. (B) Amino acid sequence identities between MF/DNase B or Spd1 and the Spd1-related putative DNases.

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