Evidence for a partial redundancy of the fibronectin‐binding proteins for the transfer of mycoloyl residues onto the cell wall arabinogalactan termini of Mycobacterium tuberculosis

Abstract

Evidence for a partial redundancy of the fibronectinbinding proteins for the transfer of mycoloyl residues onto the cell wall arabinogalactan termini of Mycobacterium tuberculosis mutant strain of M. tuberculosis restored both the cell wall-linked mycolate defect and the outer cell envelope permeability barrier property. These results are consistent with all three enzymes being involved in cell wall mycoloylation and FbpC playing a more critical role than the others or, alternatively, FbpC is able to compensate for FbpA and FbpB in ways that these enzymes cannot compensate for FbpC, pointing to a partial redundancy of Fbps. In sharp contrast, FbpD does not appear to be an active mycoloyltransferase enzyme, as it cannot complement the fbpC-inactivated mutant. Most importantly, application of Smith degradation to the cell walls of transformants demonstrated that the multiple Fbp enzymes are redundant rather than specific for the various arabinogalactan mycoloylation regions. Neither FbpA nor FbpB attaches mycoloyl residues to specific sites but, like FbpC, each enzyme transfers mycoloyl residues onto the four sites present in the arabinogalactan non-reducing end hexaarabinosides. IntroductionTuberculosis, a severe human disease, remains a major public health problem with millions of deaths annually worldwide. The aetiological agent of the disease, Mycobacterium tuberculosis, is a facultative intracellular pathogen that possesses a complex envelope of unusually low permeability, which contributes to its resistance to host defence mechanisms (Brennan and Nikaido, 1995;Draper, 1998). Schematically, going from the plasmic to the external side of the bacteria, the cell envelope is composed of a plasma membrane, a complex wall composed of a variety of covalently linked and non-covalently linked carbohydrates and lipids and an outer layer, also called a capsule, of polysaccharides and proteins with small amounts of lipids (Daffé and Draper, 1998). The mycobacterial cell wall and capsule are typified by the presence of unusual long-chain fatty acids, the so-called mycolic acids, a-alkyl b-hydroxy C60-C90 fatty acids (Barry et al., 1998;Daffé and Draper, 1998). These compounds are believed to play a central role both in the architecture of the cell wall (Brennan and Nikaido, 1995;Draper, 1998) and in the physiology of mycobacteria, their synthesis SummaryMycobacterium tuberculosis produces a series of major secreted proteins, the fibronectin-binding proteins (Fbps), also known as the antigen 85 complex, that are believed to play an essential role in the pathogenesis of tuberculosis through their mycoloyltransferase activity required for maintaining the integrity of the bacterial cell envelope. Four different fbp genes are found in the genome of M. tuberculosis, but the reason for the existence of these Fbps sharing the same substrate specificity in vitro in mycobacteria is unknown. We have shown previously that, in the heterologous host, Corynebacterium glutamicum, FbpA, FbpB and FbpC can all add mycoloyl residues to the ...

FbpA-Dependent Biosynthesis of Trehalose Dimycolate Is Required for the Intrinsic Multidrug Resistance, Cell Wall Structure, and Colonial Morphology of Mycobacterium smegmatis

“…It will be interesting to see whether disruption of this fbp gene completely eliminates TDM in the cell wall of MAR1 and leads to even higher sensitivity to antibiotics. Our in vivo results, in addition to previous reports (5,17,19,30), suggested that FbpA (and probably also FbpB) mycolyltransferase activity preferred trehalose as a substrate, while FbpC prefers arabinogalactan. These preferences may help explain the apparent redundancy of Fbp paralogs.…”

“…Curiously, resistance to the limited spectrum of antibiotics tested was unaffected. Genetic analysis showed that all three M. tuberculosis fbp genes could be disrupted individually and that they played partially redundant roles in cell wall biosynthesis (30). The fact that a synthetic analog of a Fbp substrate was able to inhibit growth and cell wall biosynthesis demonstrated that these proteins, or others having similar activities, were essential and thus attractive targets for new antimycobacterial drugs (5).…”

Enzymatic Hydrolysis of Trehalose Dimycolate Releases Free Mycolic Acids during Mycobacterial Growth in Biofilms

“…Intriguingly, although both ⌬fbpA and ⌬Msmeg_1529 mutants have comparable defects in FM synthesis, ⌬fbpA is severely defective in biofilm growth. The severity of the ⌬fbpA mutant could be due to possible defect in its cell wall biosynthesis as the gene has been previously implicated in the synthesis of mycolyl esters of cell wall (43,45).…”

The mycolyltransferase 85A, a putative drug target of Mycobacterium tuberculosis: Development of a novel assay and quantification of glycolipid-status of the mycobacterial cell wall

“…The cord factor has been suggested to be an important determinant for successful infection and survival of MTB within macrophages (Indrigo et al, 2003). Furthermore, it has been shown that 85A and 85B proteins are involved in the covalent attachment of mycolic acids to the mycobacterial cell wall (Puech et al, 2002). Several studies strengthen the hypothesis that differential expression of the antigen 85 genes may be a mechanism utilized by mycobacteria to confuse and evade the host immune system (Armitige et al, 2000;Harth et al, 2002;Ronning et al, 2004).…”