Deletion of kasB in Mycobacterium tuberculosis causes loss of acid-fastness and subclinical latent tuberculosis in immunocompetent mice

Abstract

Mycobacterium tuberculosis, the causative agent of tuberculosis, has two distinguishing characteristics: its ability to stain acid-fast and its ability to cause long-term latent infections in humans. Although this distinctive staining characteristic has often been attributed to its lipid-rich cell wall, the specific dye-retaining components were not known. Here we report that targeted deletion of kasB, one of two M. tuberculosis genes encoding distinct ␤-ketoacyl-acyl carrier protein synthases involved in mycolic acid synthesis, results in loss of acid-fast staining. Biochemical and structural analyses revealed that the ⌬kasB mutant strain synthesized mycolates with shorter chain lengths. An additional and unexpected outcome of kasB deletion was the loss of ketomycolic acid trans-cyclopropanation and a drastic reduction in methoxymycolic acid trans-cyclopropanation, activities usually associated with the trans-cyclopropane synthase CmaA2. Although deletion of kasB also markedly altered the colony morphology and abolished classic serpentine growth (cording), the most profound effect of kasB deletion was the ability of the mutant strain to persist in infected immunocompetent mice for up to 600 days without causing disease or mortality. This long-term persistence of ⌬kasB represents a model for studying latent M. tuberculosis infections and suggests that this attenuated strain may represent a valuable vaccine candidate against tuberculosis. mycolic acid ͉ Ziehl-Neelsen stain ͉ cording ͉ persistence ͉ FAS-II

Structural elucidation and genomic scrutiny of the C60–C100 mycolic acids of Segniliparus rotundus

“…HadB alone bears the characteristic 'hydratase 2 motif', representing the catalytic subunit of HadAB and HadBC heterodimers, while HadA and HadC subunits are necessary to stabilize the long acyl chain of the substrates (Sacco et al 2007). The preference of HadBC for longer substrates, as compared with HadAB, strongly suggests that HadBC is involved, as previously described for KasB, in the late elongation steps of the meromycolic chains in mycobacteria (Gao et al, 2003;Bhatt et al, 2007;Sacco et al, 2007). Protein-protein BLAST searches performed against the S. rotundus database using each of the three M. tuberculosis dehydratase subunits as a probe picked up four adjacent proteins (Table 1) -Srot2731c (180 aa), Srot2730c (142 aa), Srot2729c (183 aa) and Srot2728c (141 aa) -whose lengths are comparable with those of the M. tuberculosis Had subunits HadA (158 aa), HadB (142 aa) and HadC (166 aa).…”

“…This subtle difference could be explained by the activity of KasA when KasB is absent. A decrease in the amount of ketomycolic acid was also observed in a DkasB mutant of M. tuberculosis (Bhatt et al, 2007). In M. tuberculosis, fabD, acpM, kasA, kasB and accD6 are adjacent genes that belong to the same transcriptional unit (Fig.…”

“…The association with HadA most likely confers to HadB the ability to deal with the early elongation cycles in mycobacteria as well as in genera bearing relatively shortchain mycolic acids (Amycolicicoccus, Rhodococcus and Nocardia) and where a KasB orthologue is also absent (Table 2). This strongly suggests that HadBC is involved in the late elongation steps of the meromycolate chains in mycobacteria, as described for KasB (Gao et al, 2003;Bhatt et al, 2007). However, the existence of two different catalytic dehydratase subunits (HadB1 and HadB2 corresponding to Srot2730c and Srot2728c) with two different HadA/C (HadA Srot2731c and HadC Srot2729c) for chain specificity, is an original feature of the genome of S. rotundus.…”

iniBAC induction Is Vitamin B12- and MutAB-dependent in Mycobacterium marinum

“…To see whether a defect in lipid biosynthesis and cell envelope integrity in general would also cause induction of the operon, we transformed a kasB::tn mutant, a mutant known to produce truncated mycolic acids, with the iniBAC stress marker (23). This mutant shows reduced growth and is attenuated in zebrafish embryos.…”

Identification of Rhodococcus equi lipids recognized by host cytotoxic T lymphocytes

“…The samples were dissolved in chloroform/ methanol (2 : 1, v/v) at a concentration of 1 mg ml 21 , and 1 ml was applied directly to the sample plate, followed by addition of 1 ml 2,5-dihydroxybenzoic acid [10 mg ml 21 in chloroform/methanol (1 : 1, v/v)] as a matrix. It was analysed in the reflectron mode with an accelerating voltage operating in positive mode of 20 kV (Bhatt et al, 2007).…”