Killer Prophage for Hire

by Merry Youle

Lysogeny is a wide­spread form of sym­bio­sis among bac­teria, but hav­ing a res­i­dent prophage is a mixed bless­ing. (For a com­pre­hen­sive re­view of the plethora of propha­ges found in bac­te­ria, click here.) A prophage can bring ben­e­fits such as re­sis­tance to at­tack by other phages or novel vir­u­lence fac­tors. But along with the ben­e­fits comes a li­a­bil­ity – some­thing that we ex­ploit when we use UV or other agents to in­duce prophages to ex­cise from the host bac­te­r­ial chro­mo­some, repli­cate, and lyse the cell. Not sur­pris­ingly, bac­te­ria cap­i­tal­ize on this vulne­ra­bility. In at least one case, they use prophage in­duc­tion to elim­i­nate com­pet­ing bac­te­ria. That par­tic­u­lar case is the killing of Staphy­lo­coc­cus au­reus by the pneu­mo­coc­cus, Strep­to­coc­cus pneu­mo­niae.

Fin­ger­tips shortly af­ter a brief ex­po­sure to 35 % hy­dro­gen per­ox­ide. Source

A lit­tle back­ground first. In a pa­per pub­lished in 1922, McLeod and Gor­don re­ported that the su­per­natant from aer­o­bic pneu­mo­coc­cal cul­tures in­hib­ited the growth of staph. They iden­ti­fied the ac­tive agent as hy­dro­gen per­ox­ide (H₂O₂). Not bad for the Old Days! Since then, their ob­ser­va­tion has been cor­rob­o­rated and ex­tended by oth­ers, adding to the pic­ture of vig­or­ous com­pe­ti­tion in the heav­ily col­o­nized hu­man na­sophar­ynx.

Strep­to­coc­cus pneu­mo­niae. Source

How much H₂O₂ do pneu­mo­cocci pro­duce? Full-grown cul­tures of some strains achieve 1 mM con­cen­tra­tions. This is enough to kill some bac­te­ria and also to dam­age host tis­sues. So far only S. pneu­mo­niae and some lac­tic acid bac­te­ria are known to re­lease this much H₂O₂ into their en­vi­ron­ment. As a de­fense against H₂O₂, most bac­te­ria pro­duce cata­lase that breaks down H₂O₂. Oth­ers that lack cata­lase are highly sen­si­tive to H₂O₂ them­selves. Pneu­mo­cocci have no cata­lase. How come they sur­vive? Read on.

Like many bac­te­ria, the tar­geted staph is well de­fended against both H₂O₂ and its toxic break­down prod­ucts gen­er­ated by the in­or­ganic Fen­ton re­ac­tion. It has H₂O₂-in­ac­ti­vat­ing en­zymes (e.g., cata­lase). Yet, staph suc­cumbs to rel­a­tively low lev­els of H₂O₂. Much higher lev­els are re­quired to kill bac­te­ria by di­rect dam­age to their DNA from hy­droxyl rad­i­cals (OH•). Why is staph par­tic­u­larly vul­ner­a­ble? Here's a clue. H₂O₂ in­duces the alarm re­action known as the SOS re­sponse. In ad­di­tion to its role in mo­bi­liz­ing DNA re­pair, the SOS re­sponse in­duces res­i­dent prophages to en­ter the lytic cy­cle, thus lead­ing to cell ly­sis and death. An­other clue: staph typ­i­cally car­ries in­ducible prophages.

Putting this all to­gether, a team of re­searchers in Spain and the USA came up with an ex­pla­na­tion for how pneu­moccci kill staph. They pos­tu­lated that the low level of H₂O₂ pro­duced by the pneu­mococci trig­gers the SOS re­sponse in the staph, which in turn in­duces its prophages to go lytic, ly­sing the bac­te­ria. They treated both lyso­genic and non-lyso­genic staph strains with 0.5 mM H₂O₂. The lyso­genic strains were killed, the non-lyso­genic ones were not af­fected. The H₂O₂ treat­ment elicited an SOS re­sponse and in­duced the prophages in the lyso­genic strains. Lyso­genic mu­tants that could not mount an SOS re­sponse were not killed, and nei­ther were mu­tants whose propha­ges could not be in­duced. As an aside, the re­searchers rea­soned that lyso­genic strains of staph should be more sen­si­tive to DNA-dam­ag­ing an­tibi­otics such as ciprofloxacin that also in­duce the SOS re­sponse. In­deed, they are.

SEM mi­cro­graph of S. au­reus colonies. Source

Why, you have been won­der­ing, can pneu­moc­cci tol­er­ate H₂O₂? Even lyso­genic strains are not af­fected. They don't have cata­lase and thus would be ex­pected to have more nasty free rad­i­cals pro­duced from the H₂O₂, but appar­ent­ly they have some other, as yet un­known, pro­tec­tive mech­a­nism. And im­por­tant for our story, H₂O₂ does not trig­ger their SOS re­sponse.

This sounds like a great bac­te­ri­ci­dal tac­tic. Could we use it to treat our own staph in­fec­tions? Un­for­tu­nately, in­duc­ing prophages from staph brings un­wanted con­sequen­ces. Among other things, re­leas­ing vi­able phage into the en­vi­ron­ment could pro­mote the spread of phage-en­coded tox­ins and phage-re­lated path­o­genic­ity is­lands that en­code TSST‑1 and other su­per­anti­gen tox­ins. Too bad. As you know, staph is becom­ing in­creas­ingly re­sis­tant to an­tibi­otics (and MRSA is fre­quently in the news). This would have been a nice ad­di­tion to our ther­a­peu­tic op­tions.