RESUMEN
Four serine/threonine kinases are present in all mycobacteria: PknA, PknB, PknG and PknL. PknA and PknB are essential for growth and replication, PknG regulates metabolism, but little is known about PknL. Inactivation of pknL and adjacent regulator MSMEG_4242 in rough colony M. smegmatis mc2155 produced both smooth and rough colonies. Upon restreaking rough colonies, smooth colonies appeared at a frequency of ~ 1/250. Smooth mutants did not form biofilms, showed increased sliding motility and anomalous lipids on thin-layer chromatography, identified by mass spectrometry as lipooligosaccharides and perhaps also glycopeptidolipids. RNA-seq and Sanger sequencing revealed that all smooth mutants had inactivated lsr2 genes due to mutations and different IS1096 insertions. When complemented with lsr2, the colonies became rough, anomalous lipids disappeared and sliding motility decreased. Smooth mutants showed increased expression of IS1096 transposase TnpA and MSMEG_4727, which encodes a protein similar to PKS5. When MSMEG_4727 was deleted, smooth pknL/MSMEG_4242/lsr2 mutants reverted to rough, formed good biofilms, their motility decreased slightly and their anomalous lipids disappeared. Rough delpknL/del4242 mutants formed poor biofilms and showed decreased, aberrant sliding motility and both phenotypes were complemented with the two deleted genes. Inactivation of lsr2 changes colony morphology from rough to smooth, augments sliding motility and increases expression of MSMEG_4727 and other enzymes synthesizing lipooligosaccharides, apparently preventing biofilm formation. Similar morphological phase changes occur in other mycobacteria, likely reflecting environmental adaptations. PknL and MSMEG_4242 regulate lipid components of the outer cell envelope and their absence selects for lsr2 inactivation. A regulatory, phosphorylation cascade model is proposed.
RESUMEN
Lipids were extracted from cysticerci of the human tapeworm Taenia solium isolated from various infected pigs and analysed by two-dimensional thin-layer chromatography. These consisted of both alkali-labile and alkali-stable glycolipids, and phosphorylated non-glycosylated lipids. Because abundant and immunogenic glycolipids of parasites have been implicated in host-parasite interactions, the major lipid, an alkali-stable glycolipid, was purified by chromatography and its structure and antigenicity were determined. The structure of the major glycolipid of T. solium, GSL-I, was elucidated through a combination of chemical degradative methods, gas chromatography/mass spectrometry analyses of the degradative products, matrix-assisted-laser desorption/ionisation time of flight mass spectrometry and nuclear magnetic resonance spectroscopy. This analytical strategy led to the identification of a family of beta-galactosylceramides composed mainly of phytosphinganine (2-hydroxylated sphinganine) N-acylated by C16-C24 fatty acids, with the predominance of 2-hydroxylated homologues. Enzyme-linked immunosorbent assay showed no correlation between the antibody titres directed against GSL-I in the human sera and the infective status; in contrast, a very high specific immunoreactivity and a sensitivity above 50% were observed when GSL-I was tested with cerebrospinal fluids from well characterised infected humans. Thus, although these results do not support the use of GSL-I alone as an antigen for the detection of neurocysticercosis, its use as part of an antigen cocktail for the diagnosis of the disease in cerebrospinal fluids merits further investigations.