Weak­en­ing The My­colic Acid Fortress of M. tu­ber­cu­lo­sis

by Monika Buczek

"The earth is suf­fo­cat­ing – Swear to make them cut me open, so that I won't be buried alive."
—Fred­eric Chopin, while dy­ing of tu­ber­cu­lo­sis

Con­sum­ing the World Pop­u­la­tion

Tu­ber­cu­lo­sis (TB), is an in­fec­tious dis­ease caused by the bac­terium My­cobac­terium tu­ber­cu­lo­sis (Mtb). Symp­toms of an ac­tive TB in­fec­tion in­clude chronic cough with blood-tinged spu­tum, fever, and weight loss. In fact, prior to the 1882 dis­cov­ery of the mi­crobe by Robert Koch, TB was called "con­sump­tion" be­cause the rapid rate with which in­fected pa­tients lost body mass made them ap­pear to be "con­sumed" by some un­known en­tity. What makes TB par­tic­u­larly chill­ing is that an overtly healthy in­di­vid­ual may har­bor the mi­crobe and de­velop ac­tive dis­ease un­ex­pect­edly, then pass Mtb to oth­ers through aerosol droplets. Most in­fected per­sons do not have symp­toms, but about 10% of la­tent in­fec­tions even­tu­ally progress to ac­tive dis­ease. What it takes is a weak­ened im­mune sys­tem for the bac­te­ria to over­come it and mul­ti­ply, re­sult­ing in the pro­gres­sion from la­tent in­fec­tion to TB dis­ease.

Fig­ure 1. Anti-Tu­ber­cu­lo­sis ad­ver­tis­ing from the Ame­ri­can Lung As­so­ci­a­tion c. 1935. Just be­cause a per­son looks healthy, he/she may be in­fected with TB with­out know­ing it. Source

About one-third of the world's pop­u­la­tion has been in­fected with M. tu­ber­cu­lo­sis, with new in­fec­tions oc­cur­ring at a rate of about 1 in every 100 peo­ple each year. Al­though TB can be treated with a 6‑month reg­i­men of an­tibi­otics, drug-re­sis­tant Mtb re­mains a se­ri­ous pub­lic health is­sue in many de­vel­op­ing coun­tries. Mul­tidrug Re­sis­tant TB (MDR-TB) is re­sis­tant to the two most ef­fec­tive TB drugs: ri­fampicin and iso­ni­azid. Ex­ten­sively drug-re­sis­tant TB (EDR-TB) is re­sis­tant to these two plus three or more of the six classes of sec­ond-line drugs. To­tally drug-re­sis­tant TB (TR-TB) is re­sis­tant to all cur­rently used drugs. Where are all these drug re­sis­tant strains com­ing from? The World Health Or­ga­ni­za­tion has an idea:

"The rea­sons why mul­tidrug re­sis­tance con­tin­ues to emerge and spread are mis­man­age­ment of TB treat­ment and per­son-to-per­son trans­mis­sion. Most peo­ple with TB are cured by a strictly fol­lowed, six-month drug reg­i­men that is pro­vided to pa­tients with sup­port and su­per­vi­sion. In­ap­pro­pri­ate or in­cor­rect use of an­timi­cro­bial drugs, or use of in­ef­fec­tive for­mu­la­tions of drugs (e.g. use of sin­gle drugs, poor qual­ity med­i­cines or bad stor­age con­di­tions), and pre­ma­ture treat­ment in­ter­rup­tion can cause drug re­sis­tance, which can then be trans­mit­ted, es­pe­cially in crowded set­tings such as pris­ons and hos­pi­tals."

Ac­quir­ing high-qual­ity drugs and com­plet­ing a full 6‑month course im­poses ma­jor eco­nomic bur­dens on in­fected pa­tients, es­pe­cially those in de­vel­op­ing coun­tries. If left un­treated, about half of all cases of ac­tive TB in­fec­tion even­tu­ally re­sult in death.

Ex­ten­sive re­search is be­ing con­ducted to de­ter­mine the spe­cific vir­u­lence fac­tors that al­low Mtb to lit­er­ally root it­self within its host. Mtb has a slew of vir­u­lence fac­tors (al­though, best we know, no pro­tein en­do­tox­ins), and more are be­ing dis­cov­ered reg­u­larly. A pre­vi­ous ar­ti­cle in this blog dis­cusses one of Mtb's es­sen­tial vir­u­lence fac­tors, and its ef­fect on the host im­mune sys­tem. But Mtb not only has sep­a­rate vir­u­lence fac­tors, but ver­i­ta­ble de­fense sys­tems, one of them be­ing a vir­tu­ally im­per­me­able cell en­ve­lope.

Mycobacteria's Mi­cro­scopic Ar­mor

Fig­ure 2. Mtb "cord­ing" in liq­uid cul­ture (Acid Fast Stain, 100x mag­ni­fi­ca­tion). Source

Mtb's coat is un­usual in be­ing rich in very long and ex­otic lipids that con­sti­tute an ex­ter­nal mem­brane called the my­comem­brane. It ren­ders the bac­terium im­pen­e­tra­ble to com­po­nents of the host's im­mune sys­tem and many harsh chem­i­cals in­clud­ing strong acids as well as com­mon an­tibi­otics. Thus it plays a cru­cial role in its path­o­genic­ity. It is also in­volved in con­fer­ring re­sis­tance of Mtb to dry­ing, which aids in its trans­mis­sion be­tween per­sons. Since many of the pa­tients de­velop dis­ease in the lung, they end up cough­ing up live bac­te­ria that can per­sist for a long time in hu­man habi­ta­tions.

A re­cent re­port stud­ies fur­ther a strain of vir­u­lent Mtb that lost vir­u­lence through pas­sages on var­i­ous me­dia in the 1930's. The mu­tant strain is de­fec­tive in the syn­the­sis of my­colic acids, long chain fatty acids char­ac­ter­is­tic of the my­comem­brane. Strik­ingly, this strain shows an ob­vi­ous phe­no­typic dif­fer­ence from its vir­u­lent brother (or may be, its cousin thrice re­moved): it doesn't form the char­ac­ter­is­tic cords (Fig­ure 2) usu­ally ob­served in vir­u­lent strains. The avir­u­lent strain is still some­what in­fec­tious but can­not repli­cate within macrophages.

A ge­nomic sur­vey un­cov­ered that the avir­u­lent strain dif­fers from its par­ent in three vir­u­lence-re­lated poly­mor­phisms (SNPs). One is par­tic­u­larly in­ter­est­ing be­cause it has a nu­cleotide in­ser­tion in the hadC gene. This gene be­longs to the es­sen­tial hadA-hadB-hadC clus­ter en­cod­ing pro­teins- you guessed it- HadA, HadB, and HadC. These are sub­units of two het­erodimeric de­hy­dratases, HadAB, and HadBC and part of Mtb's fatty acid syn­thase type II (FAS-II) sys­tem. This sys­tem is re­spon­si­ble for elon­gat­ing fatty acids and is in­volved in the biosyn­the­sis of the my­colic acids, which are long-chain α‑alkyl β‑hydroxy fatty acids con­tain­ing be­tween 74–89 car­bons. (To read about the other two SNPs please click here for an ar­ti­cle about a mu­ta­tion in the tran­scrip­tional reg­u­la­tor PhoP, and here for an ar­ti­cle about a mu­ta­tion dis­rupt­ing ph­thio­cerol dimy­co­cerosate (PDIM) and gly­co­sylphe­nol-PDIM biosyn­the­sis path­way.)

Fig­ure 3. Biofilm pro­duc­tion and cord­ing in Mtb. Strains H37Ra and H37Rv are avir­u­lent and vir­u­lent respec­tively iso­lated from the same host (pan­els 1 and 3). A func­tional wt copy of hadC was in­tro­duced into the avir­u­lent strain de­noted as "H37Ra/hadCrv" (panel 2). A dele­tion of hadC within the vir­u­lent strain is de­noted as "H37RvΔhadC" (panel 4) and when func­tional hadC is re-in­tro­duced into this strain it is de­noted as "H37RvΔhadC/ had­CRv" (panel 5). Source

These pe­cu­liar waxy com­pounds are ma­jor com­po­nents of the my­comem­brane and are re­spon­si­ble for sta­bi­liz­ing its ar­chi­tec­ture and de­creas­ing its per­me­abil­ity. Thus, my­colic acids con­tribute sig­nif­i­cantly to the vir­u­lence and per­sis­tence of the strain within a host. They are re­spon­si­ble, among other things, for the sur­vival fo the bac­te­ria within macrophages. Ac­cord­ingly, the au­thors set out to de­ter­mine if there might be a link be­tween mu­ta­tions in hadC and at­ten­u­a­tion of Mtb vir­u­lence.

Com­pro­mis­ing Mtb's de­fenses

As any bac­te­r­ial ge­neti­cist would, the au­thors first re­moved the hadC gene from a vir­u­lent strain of Mtb in or­der to pin­point phe­no­typic changes in the mu­tant. Dele­tion of hadC not only de­creased the strain's vir­u­lence, but also re­duced acid fast­ness and ren­dered it more sen­si­tive to di­verse com­pounds, par­tic­u­larly the large and lipophilic an­tibi­otic ri­fampin. It is likely then, that the mu­ta­tion dis­rupts the or­ga­ni­za­tion of the my­comem­brane.

It has been known that the struc­ture of the my­comem­brane is in­volved in Mtb ag­gre­gat­ing into biofilms or into plank­tonic struc­tures known as cords. The au­thors an­a­lyzed mu­tant and wild type hadC Mtb strains for colony mor­phol­ogy and cord­ing abil­ity, as well as de­ter­min­ing their vir­u­lence by mea­sur­ing spleen size in in­fected se­verely im­mun­od­e­fi­cient (SCID) mice. Re­moval of hadC ac­tiv­ity ren­dered a vir­u­lent strain in­ca­pable of both biofilm pro­duc­tion as well as cord­ing abil­ity. Fur­ther­more, re-in­tro­duc­ing a func­tional copy of hadC into ei­ther the avir­u­lent strain or a hadC dele­tion strain re­gained both ca­pa­bil­i­ties (Fig­ures 3 and 4).

A new tar­get for an­tibi­otic re­sis­tant tu­ber­cu­lo­sis: HadC

Fig­ure 4. Spleens in­fected by avir­u­lent H37Ra and H37RvΔhadC are much smaller than those in­fected by the vir­u­lent strains H37Rv, H37Ra/hadCRv and H37RvΔhadC/ had­Crv. Source

At the end of the day, hadC is ob­vi­ously an im­por­tant gene for Mtb in­fec­tion, be­ing re­spon­si­ble for loss of vir­u­lence and cord­ing mor­phol­ogy. This is most likely due to im­por­tant changes in the struc­ture of my­colic acids in the my­comem­brane, which nor­mally cre­ates a vir­tual fortress against all at­tacks by the host, be they by the im­mune sys­tem or by an­tibi­otics. The iden­ti­fi­ca­tion of a ge­netic mu­ta­tion that ren­ders Mtb vul­ner­a­ble to ex­ter­nal com­pounds is ex­tremely im­por­tant be­cause it pro­vides us with a new at­trac­tive tar­get for drug ther­apy that may make an im­pen­e­tra­ble bac­terium per­me­able to a slew of drugs that pre­vi­ously were use­less. This ini­tial work kin­dles the hope that we are not doomed to a world of to­tally an­tibi­otic re­sis­tant tu­ber­cu­lo­sis.

 

Monika Buczek is a Ph.D. can­di­date in the Mol­e­c­u­lar, Cel­lu­lar and De­vel­op­men­tal Bi­ol­ogy De­part­ment at the City Uni­ver­sity of New York Grad­u­ate Cen­ter. She is a mem­ber of Dr. Anu­rad­ha Janakiraman's lab at the CCNY Cen­ter for Dis­cov­ery and In­no­va­tion and her the­sis re­search fo­cuses on the pro­te­olytic reg­u­la­tion of E.coli cy­toki­ne­sis. She also teaches as an ad­junct lec­turer in Mol­e­c­u­lar bi­ol­ogy and Mi­cro­bi­ol­ogy at the City Col­lege of New York.