Inactivation of tesA reduces cell wall lipid production and increases drug susceptibility in mycobacteria.

Phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) are structurally related lipids noncovalently bound to the outer cell wall layer of Mycobacterium tuberculosis, Mycobacterium leprae, and several opportunistic mycobacterial human pathogens. PDIMs and PGLs are important effectors of virulence. Elucidation of the biosynthesis of these complex lipids will not only expand our understanding of mycobacterial cell wall biosynthesis, but it may also illuminate potential routes to novel therapeutics against mycobacterial infections. We report the construction of an in-frame deletion mutant of tesA (encoding a type II thioesterase) in the opportunistic human pathogen Mycobacterium marinum and the characterization of this mutant and its corresponding complemented strain control in terms of PDIM and PGL production. The growth and antibiotic susceptibility of these strains were also probed and compared with the parental wild-type strain. We show that deletion of tesA leads to a mutant that produces only traces of PDIMs and PGLs, has a slight growth yield increase and displays a substantial hypersusceptibility to several antibiotics. We also provide a robust model for the three-dimensional structure of M. marinum TesA (TesAmm) and demonstrate that a Ser-to-Ala substitution in the predicted catalytic Ser of TesAmm renders a mutant that recapitulates the phenotype of the tesA deletion mutant. Overall, our studies demonstrate a critical role for tesA in mycobacterial biology, advance our understanding of the biosynthesis of an important group of polyketide synthase-derived mycobacterial lipids, and suggest that drugs aimed at blocking PDIM and/or PGL production might synergize with antibiotic therapy in the control of mycobacterial infections.

Do nerve growth factor-related mechanisms contribute to loss of cutaneous nociception in leprosy?

While sensory loss in leprosy skin is the consequence of invasion by M. leprae of Schwann cells related to unmyelinated fibres, early loss of cutaneous pain sensation, even in the presence of nerve fibres and inflammation, is a hallmark of leprosy, and requires explanation. In normal skin, nerve growth factor (NGF) is produced by basal keratinocytes, and acts via its high affinity receptor (trk A) on nociceptor nerve fibres to increase their sensitivity, particularly in inflammation. We have therefore studied NGF- and trk A-like immunoreactivity in affected skin and mirror-site clinically-unaffected skin from patients with leprosy, and compared these with non-leprosy, control skin, following quantitative sensory testing at each site. Sensory tests were within normal limits in clinically-unaffected leprosy skin, but markedly abnormal in affected skin. Sub-epidermal PGP 9.5- and trk A- positive nerve fibres were reduced only in affected leprosy skin, with fewer fibres contacting keratinocytes. However, NGF-immunoreactivity in basal keratinocytes, and intra-epidermal PGP 9.5-positive nerve fibres, were reduced in both sites compared to non-leprosy controls, as were nerve fibres positive for the sensory neurone specific sodium channel SNS/PN3, which is regulated by NGF, and may mediate inflammation-induced hypersensitivity. Keratinocyte trk A expression (which mediates an autocrine role for NGF) was increased in clinically affected and unaffected skin, suggesting a compensatory mechanism secondary to reduced NGF secretion at both sites. We conclude that decreased NGF- and SNS/PN3-immunoreactivity, and loss of intra-epidermal innervation, may be found without sensory loss on quantitative testing in clinically-unaffected skin in leprosy; this appears to be a sub-clinical change, and may explain the lack of cutaneous pain with inflammation. Sensory loss occurred with reduced sub-epidermal nerve fibres in affected skin, but these still showed trk A-staining, suggesting NGF treatment may restore pain sensation.

Correlation of quantitative tests of nerve and target organ dysfunction with skin immunohistology in leprosy.

Loss of nociception and hypohidrosis in skin are hallmarks of leprosy, attributed to early invasion by Mycobacterium leprae of Schwann cells related to unmyelinated nerve fibres. We have studied skin lesions and contralateral clinically unaffected skin in 28 patients across the leprosy spectrum with a range of selective quantitative sensory and autonomic tests, prior to biopsy of both sites. Unaffected sites showed normal skin innervation, when antibodies to the pan-neuronal marker PGP (protein gene product) 9.5 were used, with the exception of intraepidermal fibres which were not detected in the majority of cases. Elevation of thermal thresholds and reduced sensory axon-reflex flare responses in affected skin correlated with decreased nerve fibres in the subepidermis, e.g. axon-reflex flux units (means+/-SEM) for no detectable innervation; decreased innervation; and clinically unaffected skin, were 23+/-3.1; 41.2+/-7.3; and 84.5+/-4.0, respectively. Reduced nicotine-induced axon-reflex sweating was correlated with decreased innervation of sweat glands. Where methacholine-induced direct activation of sweat glands was affected, there was inflammatory infiltrate and loss of sweat gland structure. This study demonstrates a correlation between selective nerve dysfunction on clinical tests and morphological changes in skin, irrespective of the type of leprosy, and is the first to show that loss of sweating in leprosy may result either from decreased innervation and/or involvement of the sweat glands. The findings have implications for the selection and monitoring of patients with leprosy in clinical trials which aim to restore cutaneous function.