The emb genes are conserved among different mycobacteria. In Mycobacterium smegmatis and Mycobacterium tuberculosis, they belong to an operon comprising three genes, embC, embA, and embB. The EmbB protein has been proposed to be the target of ethambutol, a drug which is known to inhibit the synthesis of the arabinan portion of the mycobacterial cell wall arabinogalactan (AG). To further define the role of EmbB protein in arabinan biosynthesis, embA, -B, and -C genes were inactivated individually by homologous recombination in M. smegmatis. All three mutants were viable, and among the three, the slowest growing embB ؊ mutant encountered profound morphological changes and exhibited a higher sensitivity to hydrophobic drugs and detergents, presumably due to an increase in cell wall permeability. Furthermore, chemical analyses showed that there was a diminution in the arabinose content of arabinogalactan from the embA ؊ and embB ؊ mutants. Specifically, in comparison with the wild-type strain, the crucial terminal hexaarabinofuranosyl motif, which is a template for mycolylation, was altered in both embA ؊ and embB

The unique terminal arabinan motifs of mycobacterial lipoarabinomannan (LAM), which are mannose-capped to different extents, probably constitute the single most important structural entity engaged in receptor binding and subsequent immunopathogenesis. We have developed a concerted approach of endoarabinanase digestion coupled with chromatography and mass spectrometry analysis to rapidly identify and quantitatively map the complement of such terminal units among the clinical isolates of different virulence and drug resistance profiles. In comparison with LAM from laboratory strains of Mycobacterium tuberculosis, an ethambutol (Emb) resistant clinical isolate was shown to have a significantly higher proportion of nonmannose capped arabinan termini. More drastically, the mannose capping was completely inhibited when an Emb-susceptible strain was grown in the presence of subminimal inhibitory concentration of Emb. Both cases resulted in an increase of arabinose to mannose ratio in the overall glycosyl composition of LAM. Emb, therefore, not only could affect the complete elaboration of the arabinan as found previously for LAM from Mycobacterium smegmatis resistant mutant but also could inhibit the extent of mannose capping and hence its associated biological functions in M. tuberculosis. Unexpectedly, an intrinsically Emb-resistant Mycobacterium avium isolate of smooth transparent colony morphology was found to have most of its arabinan termini capped with a single mannose residue instead of the more common dimannoside as established for LAM from M. tuberculosis. This is the first report on the LAM structure from M. avium complex, an increasingly important opportunistic infectious agent afflicting AIDS patients.One of the most prominent macromolecular entities of all mycobacterial cell walls is the arabinan, a common constituent of both the arabinogalactan (AG) 1 and lipoarabinomannan (LAM) (1). In the chemical setting of mycolylarabinogalactanpeptidoglycan complex, AG forms an integral part of the cell wall proper, whereas the wall associated LAM has been implicated as a key molecule involved in the virulence and immunopathogenesis of tuberculosis (2). The arabinan of LAM is attached to a mannan core that extends from a phosphatidylinositol mannoside anchor at the reducing end. Of major biological significance is the degree and chemical nature of capping functions and other substituents on the arabinan. It was demonstrated that the arabinan chains of LAMs from Mycobacterium tuberculosis, as well as the attenuated Mycobacterium bovis BCG vaccine strain and Mycobacterium leprae, the etiological agent of leprosy, are mannose-capped to varying degrees (40 -70%). In contrast, the majority of the arabinan termini from the rapidly growing, noninfective Mycobacterium smegmatis are uncapped, whereas a minor portion terminates with inositol phosphate (3-6). The mannose-capped LAM isolated from M. tuberculosis is generally less effective at stimulating macrophages than the non-mannose-capped LAM from fast growing strains (2),...

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Summary The reproductive‐derived serine protease inhibitor Kazal‐type (Spink) has been identified in seminal plasma, and Spink–spermatozoa binding has been illustrated in many mammalian species including human. We used mice as experimental animal to study the mode of Spink action in the modulation of mammalian sperm activity. A Spink3‐binding zone was cytochemically stained on the sperm head at apical hook separated from intact acrosome, whether the cells were capacitated or not. The Spink3–spermatozoa binding neither changed the population of cells in the uncapacitated, capacitated and acrosome‐reacted status nor affected the capacitation‐related protein phosphorylation and cell motility enhancement. Despite that, the Spink–spermatozoa interaction resulted in decreasing the intracellular calcium concentration ([Ca2+]i) of the cell head and suppressing both the acrosome reaction induced by Ca+2 ionophore A23187 and the cell fertility. Furthermore, Spink3 seen on the head of spermatozoa in the uterine cavity after coitus could be removed by the trypsin‐like activity in the uterine fluid of oestrous females, and free Spink3 in the uterine cavity suppressed the protease activity. We integrated our data to shed light on the molecular mechanism of how Spink and its inhibiting protease are interplayed to modulate the activity of mammalian spermatozoa during their transit in the reproductive tract.

The molecular basis of mammalian sperm capacitation, either in vivo in the female reproductive tract, or in vitro, is poorly understood. It is well known that sperm capacitation is associated with an increase in tyrosine phosphorylation of a subset of proteins. We resolved the phosphoproteins in the cell lysate of mouse sperm after capacitation by 2-DE. One tyrosine-phosphorylated 130-kDa spot was trypsin-digested, and six oligopeptide sequences were established from the MS data. These were confirmed in a CCCTC-binding nuclear factor (CTCF), a widely expressed and highly conserved protein. Further, both an anti-phosphotyrosine antibody and an anti-CTCF antibody showed immunoreactivity to a 130-kDa component in the immunoprecipitates obtained after incubation of the cell lysate from the capacitated sperm using another anti-CTCF antibody. The data support the presence of a tyrosine-phosphorylated CTCF in the capacitated sperm. Immunolocalization of the CTCF revealed fluorescent staining in the acrosome region in both capacitated and incapacitated sperm. The electrophoretic mobility shift assay, using a CTCF target sequence 5'-GGCGGCGCCGCTAGGGGTCTCTCT-3' found in the promoter of the amyloid beta-protein precursor, manifested that, relative to CTCF in the incapacitated sperm, the tyrosine-phosphorylated protein in the capacitated sperm had stronger affinity to the CTCF target sequence.