Why Lis­te­ria Is Com­pe­tent to Be Vir­u­lent

by S. Mar­vin Fried­man

It is down­right scan­dalous that in our hi-tech world food-borne in­fec­tions should be so preva­lent (some 48 mil­lion cases a year in the US alone, with about 3000 deaths). The tools to take care of these prob­lems are hardly mys­te­ri­ous, re­quir­ing mainly safe food pro­duc­tion and preser­va­tion. High on the list of bac­te­r­ial of­fend­ers is Lis­te­ria mono­cy­to­genes, a Gram-pos­i­tive fac­ul­ta­tive in­tra­cel­lu­lar pathogen that is ac­quired through in­ges­tion of con­t­a­m­i­nated food or flu­ids. Lis­te­ria is found in un­cooked meats, veg­eta­bles, fruits such as can­taloupes, un­pas­teur­ized milk and milk prod­ucts, and some processed foods. Al­though pas­teur­iza­tion and suf­fi­cient cook­ing will kill Lis­te­ria, con­t­a­m­i­na­tion of­ten oc­curs af­ter cook­ing and be­fore pack­ag­ing. Lis­te­rio­sis is a dis­ease pri­mar­ily af­fect­ing preg­nant women, new­borns, adults with com­pro­mised im­mune sys­tems, and the el­derly. The two most com­mon life-threat­en­ing symp­toms are sep­sis and menin­gi­tis. This is a se­ri­ous dis­ease with a rel­a­tively high mor­tal­ity that can reach 25%.

A lis­te­rio­sis out­break of 2011 caused 147 cases of ill­ness and 33 deaths in 28 states in the US. The sourcewas can­taloupes grown at one Col­orado farm. Source

L. mono­cy­to­genes is an in­tra­cel­lu­lar par­a­site of ep­ithe­lial cells and macrophages. It ini­tially re­sides in a mem­brane-bound vac­uole, the phago­so­mal vesi­cle, which is a dan­ger­ous site for many bac­te­ria. In or­der to carry out a suc­cess­ful in­fec­tion, it es­capes into the host cell's cy­to­plasm by lysing the vac­uo­lar mem­brane via a pore-form­ing he­molysin, lis­te­ri­olysin and two phos­pho­li­pases. How­ever, the pre­cise mech­a­nism of this es­cape is not fully un­der­stood. Once within the host cell cy­to­plasm, the bac­te­ria repli­cate and use the host's actin fil­a­ment net­work to pro­pel them­selves within the cell and from cell to cell.

A schematic draw­ing of the ac­ti­va­tion of Lis­te­ria pro­teins that al­low it to es­cape form the phago­some. Source

A re­cent re­port tells us that there is more to the es­cape story and that an­other sys­tem is also in­volved. This one, it turns out, con­sists of three oper­ons (comG, comF, and comE) that are ho­mol­o­gous to the ones used by Bacil­lus sub­tilis to es­tab­lish com­pe­tence, the abil­ity to read­ily take up DNA that can lead to ge­netic trans­for­ma­tion. This com­plex com­pe­tence sys­tem has been ex­ten­sively stud­ied in B. sub­tilis and other nat­u­rally com­pe­tent bac­te­ria such as Strep­to­coc­cus pneu­mo­niae. Lis­te­ria pos­sesses a num­ber of the so-called late com genes some of which, as this pa­per re­veals, are es­sen­tial for es­cape from the phago­so­mal vesi­cle.

The story is in­ter­est­ing, among other things, be­cause it il­lus­trates how a com­plex ge­netic sys­tem di­verged to al­low quite dif­fer­ent func­tions. The two func­tions dis­cussed here, com­pe­tence in some bac­te­ria and es­cape from vac­uoles in oth­ers, may well have evolved from a com­mon an­ces­tral bac­terium where, for all we know, it may have been im­pli­cated in yet dif­fer­ent func­tions. Ac­tu­ally, up­take of DNA and dam­ag­ing the vacuole's mem­brane have in com­mon that they both al­ter cell sur­face prop­er­ties, dis­parate though the two sys­tems may be. Of in­ter­est is that Lis­te­ria, al­though en­dowed with quite a few of the com genes, does not pos­sess cer­tain ones known to be di­rectly re­lated to DNA up­take in B. sub­tilis. It's no won­der that re­peated ef­forts to trans­form Lis­te­ria, even strains con­tain­ing an in­tact comK gene, have al­ways failed.

L. mono­cy­to­genes Com­pe­tence Genes Are In­duced dur­ing In­tra­cel­lu­lar Growth (A) Mi­croar­ray analy­sis of gene ex­pres­sion in bac­te­ria grown in­tra­cel­lu­larly in macrophage cells for 6 hr rel­a­tive to gene ex­pres­sion in bac­te­ria in mid-ex­po­nen­tial growth in Brain Heart Infusion(BHI) medium. (B) RT-qPCR analy­sis of late com genes tran­scrip­tion lev­els upon in­tra­cel­lu­lar growth in macrophage cells for 6 hr and dur­ing mid-ex­po­nen­tial growth in BHI medium. Tran­scrip­tion lev­els are rep­re­sented as rel­a­tive quan­tity (RQ), in­tra­cel­lu­lar ver­sus BHI medium growth. Source

This study shows that the com sys­tem is es­sen­tial for path­o­genic­ity. Us­ing mi­croar­rays, the au­thors found that the late com genes in all three oper­ons are strongly in­duced upon in­fec­tion of macrophages. Em­ploy­ing dele­tion mu­tants, the au­thors showed that only comG pro­teins (in­volved in mak­ing a Type II pro­tein-se­cre­tion pseudopilus)and comEC (a trans­mem­brane chan­nel pro­tein) were re­quired for in­tra­cel­lu­lar growth of Lis­te­ria both in macrophage­sand in mice (but not for growth in broth). These two genes are not nec­es­sary for repli­ca­tion of the bac­te­ria in the cy­tosol, sug­gest­ing fur­ther that they may play a role ear­lier on, namely in phago­so­mal es­cape. How did they find this out? Cy­toso­lic wild type Lis­te­ria nu­cle­ate actin fil­a­ments but mu­tants in comG and comEC do not, telling us that they had not es­caped from the phago­somes.

The Com pro­teins re­quired for vesi­cle es­cape are un­der the con­trol of a sole reg­u­la­tory pro­tein, comK. This pro­tein car­ries a big load on its shoul­ders (by con­trast, B. sub­tilis and S. pneu­mo­niae have sev­eral Com pro­tein reg­u­la­tors). The comK gene it­self is un­der an in­ter­est­ing con­trol mech­a­nism. In grow­ing Lis­te­ria, this gene is not func­tional be­cause it is in­ter­rupted by a tem­per­ate prophage but it comes to life when the or­gan­ism in­fects host cells. The prophage is now pre­cisely ex­cised by in­te­grase, leav­ing the two ends of the comK gene to unite to make a func­tional pro­tein. Such prophage-ex­ci­sion mech­a­nisms to reg­u­late gene ex­pres­sion have been re­ported in B. sub­tilis, S. pneu­mo­niae, and a few other species. Mu­tants with dele­tions of the comK 5'- or 3' ter­mini or lack­ing the en­tire in­te­grase gene (int) grew like wild type bac­te­ria in broth but were not ca­pa­ble of growth in macrophages. This phe­no­type was re­versed by in­tro­duc­ing a plas­mid con­tain­ing ei­ther an in­tact int or a comK gene, sug­gest­ing that phage ex­ci­sion is specif­i­cally in­volved in pro­duc­ing an in­tact comK gene.

Un­like other sys­tems where the prophages in­volved in ex­ci­sion are cryp­tic or rem­nants of a phage, this Lis­te­ria prophage ac­tu­ally en­codes for an ac­tive, vi­able phage. Sur­pris­ingly, a plaque as­say re­vealed that al­though the prophage had been ex­cised from the genome, the usual en­su­ing se­quence of phage prop­a­ga­tion and re­lease of ma­ture viri­ons was blocked in the in­fected macrophages. Why phage mat­u­ra­tion is aborted here is not yet known, nor is the sig­nal that ac­ti­vates the in­te­grase gene within the phago­so­mal vesi­cle. It would seem like a good thing for Lis­te­ria to have a mech­a­nism to in­hibit lytic phage for­ma­tion, but what is it? Some­thing about the con­di­tions within the vac­uole maybe?

This pa­per re­veals a unique con­trol mech­a­nism con­tribut­ing to vir­u­lence in L. mono­cy­to­genes. Some as yet un­known sig­nal within the phago­some trig­gers ex­ci­sion of the prophage from the bac­te­r­ial genome, lib­er­at­ing an in­tact and func­tional tran­scrip­tion­al­reg­u­la­tor that al­lows ex­pres­sion of pro­teins from the three com oper­ons. This leads to the for­ma­tion of two com­po­nents in­volved in phago­so­mal es­cape. Exit from the phago­some into the host cell cy­tosol is of crit­i­cal im­por­tance and is re­quired for a suc­cess­ful in­fec­tion by Lis­te­ria. Once again, a path­o­genic bac­terium demon­strates its abil­ity to se­lect evolv­ing path­ways that al­low them to fine-tune dis­ease pro­duc­tion.

Ref­er­ences

Ra­bi­novich L, Si­gal N, Boro­vok I, Nir-Paz R, Her­skovits AA (2012). Prophage ex­ci­sion ac­ti­vates Lis­te­ria com­pe­tence genes that pro­mote phago­so­mal es­cape and vir­u­lence. Cell, 150 (4), 792−802. PMID 22901809

Scott J, Thomp­son-May­berry P, Lah­mamsi S, King CJ, Mc­Shan WM (2008). Phage-as­so­ci­ated mu­ta­tor phe­no­type in group A strep­to­coc­cus. Jour­nal of Bac­te­ri­ol­ogy, 190 (19), 6290−6301. PMID 18676670

Kunkel B, Losick R, Stragier P (1990). The Bacil­lus sub­tilis gene for the de­vel­op­ment tran­scrip­tion fac­tor sigma K is gen­er­ated by ex­ci­sion of a dis­pens­able DNA el­e­ment con­tain­ing a sporu­la­tion re­com­bi­nase gene. Genes & de­vel­op­ment, 4 (4), 525−535. PMID 2163341

Mar­vin is Pro­fes­sor Emer­i­tus in the De­part­ment of Bi­o­log­i­cal Sci­ences at Hunter Col­lege of CUNY in New York City, and an As­so­ciate Blog­ger for Small Things Con­sid­ered.