Surface spreading motility shown by a group of phylogenetically related, rapidly growing pigmented mycobacteria suggests that motility is a common property of mycobacterial species but is restricted to smooth colonies

Motility in mycobacteria was described for the first time in 1999. It was reported that Mycobacterium smegmatis and Mycobacterium avium could spread on the surface of solid growth medium by a sliding mechanism and that the presence of cell wall glycopeptidolipids was essential for motility. We recently reported that Mycobacterium vaccae can also spread on growth medium surfaces; however, only smooth colonies presented this property. Smooth colonies of M. vaccae do not produce glycopeptidolipids but contain a saturated polyester that is absent in rough colonies. Here, we demonstrate that Mycobacterium chubuense, Mycobacterium gilvum, Mycobacterium obuense, and Mycobacterium parafortuitum, which are phylogenetically related to M. vaccae, are also motile. Such motility is restricted to smooth colonies, since natural rough mutants are nonmotile. Thin-layer chromatography analysis of the content of cell wall lipids confirmed the absence of glycopeptidolipids. However, compounds like the above-mentioned M. vaccae polyester were detected in all the strains but only in smooth colonies. Scanning electron microscopy showed great differences in the arrangement of the cells between smooth and rough colonies. The data obtained suggest that motility is a common property of environmental mycobacteria, and this capacity correlates with the smooth colonial morphotype. The species studied in this work do not contain glycopeptidolipids, so cell wall compounds or extracellular materials other than glycopeptidolipids are implicated in mycobacterial motility. Furthermore, both smooth motile and rough nonmotile variants formed biofilms on glass and polystyrene surfaces.     

Identification of a peptide synthetase involved in the biosynthesis of glycopeptidolipids of Mycobacterium smegmatis

Five rough colony mutants of Mycobacterium smegmatis mc2155 were produced by transposon mutagenesis. The mutants were unable to synthesize glycopeptidolipids that are normally abundant in the cell wall of wild-type M. smegmatis. The glycopeptidolipids have a lipopeptide core comprising a fatty acid amide linked to a tetrapeptide that is modified with O-methylated rhamnose and O-acylated 6-deoxy talose. Compositional analysis of lipids extracted from the mutants indicated that the defect in glycopeptidolipid synthesis occurred in the assembly of the lipopeptide core. No other defects or compensatory changes in cell wall structure were detected in the mutants. All five mutants had transposon insertions in a gene encoding an enzyme belonging to the peptide synthetase family. Targeted disruption of the gene in the wild-type strain gave a phenotype identical to that of the five transposon mutants. The M. smegmatis peptide synthetase gene is predicted to encode four modules that each contain domains for cofactor binding and for amino acid recognition and adenylation. Three modules also have amino acid racemase domains. These data suggest that the common lipopeptide core of these important cell wall glycolipids is synthesized by a peptide synthetase.     

Sliding motility in mycobacteria

Mycobacteria are nonflagellated gram-positive microorganisms. Previously thought to be nonmotile, we show here that Mycobacterium smegmatis can spread on the surface of growth medium by a sliding mechanism. M. smegmatis spreads as a monolayer of cells which are arranged in pseudofilaments by close cell-to-cell contacts, predominantly along their longitudinal axis. The monolayer moves away from the inoculation point as a unit with only minor rearrangements. No extracellular structures such as pili or fimbriae appear to be involved in this process. The ability to translocate over the surface correlates with the presence of glycopeptidolipids, a mycobacterium-specific class of amphiphilic molecules located in the outermost layer of the cell envelope. We present evidence that surface motility is not restricted to M. smegmatis but is also a property of the slow-growing opportunistic pathogen M. avium. This form of motility could play an important role in surface colonization by mycobacteria in the environment as well as in the host.     

Proteomics and mass spectrometric studies reveal planktonic growth of Mycobacterium smegmatis in biofilm cultures in the absence of rpoZ

Mycobacterium smegmatis is known to form biofilms and many cell surface molecules like core glycopeptidolipids and short-chain mycolates appear to play important role in the process. However, the involvement of the cell surface molecules in mycobacteria towards complete maturation of biofilms is still not clear. This work demonstrates the importance of the glycopeptidolipid species with hydroxylated alkyl chain and the epoxylated mycolic acids, during the process of biofilm development. In our previous study, we reported the impairment of biofilm formation in rpoZ-deleted M. smegmatis, where rpoZ codes for the omega subunit of RNA polymerase (R. Mathew, R. Mukherjee, R. Balachandar, D. Chatterji, Microbiology 152 (2006) 1741). Here we report the occurrence of planktonic growth in a mc(2)155 strain which is devoid of rpoZ gene. This strain is deficient in selective incorporation of the hydroxylated glycopeptidolipids and the epoxy mycolates to their respective locations in the cell wall. Hence it forms a mutant biofilm defective in maturation, wherein the cells undertake various alternative metabolic pathways to survive in an environment where oxygen, the terminal electron acceptor, is limiting.     

Cell wall structure of a mutant of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids

A mutant strain of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids was recently isolated (Liu, J., and Nikaido, H. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4011-4016). This mutant failed to synthesize full-length mycolic acids and accumulated a series of long chain beta-hydroxymeromycolates. In this work, we provide a detailed characterization of the localization of meromycolates and of the cell wall structure of the mutant. Thin layer chromatography showed that the insoluble cell wall matrix remaining after extraction with chloroform/methanol and SDS still contained a large portion of the total meromycolates. Matrix-assisted laser desorption/ionization and electrospray ionization mass spectroscopy analysis of fragments arising from Smith degradation of the insoluble cell wall matrix revealed that the meromycolates were covalently attached to arabinogalactan at the 5-OH positions of the terminal arabinofuranosyl residues. The arabinogalactan appeared to be normal in the mutant strain, as analyzed by NMR. Analysis of organic phase lipids showed that the mutant cell wall contained some of the extractable lipids but lacked glycopeptidolipids and lipooligosaccharides. Differential scanning calorimetry of the mutant cell wall failed to show the large cooperative thermal transitions typical of intact mycobacterial cell walls. Transmission electron microscopy showed that the mutant cell wall had an abnormal ultrastructure (without the electron-transparent zone associated with the asymmetric mycolate lipid layer). Taken together, these results demonstrate the importance of mycolic acids for the structural and functional integrity of the mycobacterial cell wall. The lack of highly organized lipid domains in the mutant cell wall explains the drug-sensitive and temperature-sensitive phenotypes of the mutant.     

Inactivation of lsr2 results in a hypermotile phenotype in Mycobacterium smegmatis

Mycobacterial species are characterized by the presence of lipid-rich, hydrophobic cell envelopes. These cell envelopes contribute to properties such as roughness of colonies, aggregation of cells in liquid culture without detergent, and biofilm formation. We describe here a mutant strain of Mycobacterium smegmatis, called DL1215, which demonstrates marked deviations from the above-mentioned phenotypes. DL1215 arose spontaneously from a strain deficient for the stringent response (M. smegmatis Delta rel(Msm) strain) and is not a reversion to a wild-type phenotype. The nature of the spontaneous mutation was a single base-pair deletion in the lsr2 gene, leading to the formation of a truncated protein product. The DL1215 strain was complicated by having both inactivated rel(Msm) and lsr2 genes, and so a single lsr2 mutant was created to analyze the gene's function. The lsr2 gene was inactivated in the wild-type M. smegmatis mc(2)155 strain by allelic replacement to create strain DL2008. Strain DL2008 shows characteristics unique from those of both the wild-type and Delta rel(Msm) strains, some of which include a greatly enhanced ability to slide over agar surfaces (referred to here as "hypermotility"), greater resistance to phage infection and to the antibiotic kanamycin, and an inability to form biofilms. Complementation of the DL2008 mutant with a plasmid containing lsr2 (pLSR2) reverts the strain to the mc(2)155 phenotype. Although these phenotypic differences allude to changes in cell surface lipids, no difference is observed in glycopeptidolipids, polar lipids, apolar lipids, or mycolic acids of the cell wall.     

Structural and functional alterations of lipid-depleted ascites tumor cells in culture

Ascites tumor cells growth-arrested in lipid-depleted medium were modified with respect to their lipid composition, i.e. mainly cholesterol and the phospholipid fraction. These so-called lipid-depleted cells were generally smaller, had a surface area reduced by 55% compared to the control cells and had an altered cell surface architecture with large parts being smooth, interrupted by isolated bundles of microvilli and blebs as revealed by scanning electron microscopy. This deorganization process of the cells. Lectin-induced agglutination and receptor binding capacity was reduced, and also the receptor distribution was changed resulting in a cap-like formation on the surface as shown with FITC-labelled concanavalin A. The reduction in lipid content yielding a lower C/P ratio profoundly decreased the plasma membrane fluidity which was determined by fluorescence polarization measurements. Studies on fatty acid and cholesterol de novo synthesis revealed only small increases under lipid-free conditions not sufficient to meet the requirements of the lipid-depleted cells for these substances. It is therefore concluded that ascites tumor cells need exogenous preformed lipids for adequate functioning of the cell.     

Glycopeptidolipids from Mycobacterium fortuitum: a variant in the structure of C-mycoside

Strains from the Mycobacterium fortuitum complex contain surface species-specific lipids allowing their precise identification. In M. fortuitum biovar. peregrinum two major glycopeptidolipids, of the C-mycoside type, were characterized by a combination of chemical analyses, NMR, and FAB mass spectrometry. Important information was obtained by mass spectrometry both on their molecular weight and on the peptide and saccharide sequences without any derivatization. The basic structure of the two compounds was shown to be [formula: see text] The disaccharide part linked O-glycosidically to alaninol was either 3,4-di-O-methyl-alpha-L-rhamnopyranosyl (1----2) 3,4-di-O-methyl-alpha-L-rhamnopyranoside (mycoside I) or 3-O-methyl-alpha-L-rhamnopyranosyl (1----2) 3,4-di-O-methyl-alpha-L-rhamnopyranoside (mycoside II). This is an unusual structure of a C-mycoside since neither 6-deoxytalose nor its derivatives are present. Moreover, the oligosaccharide part is linked to the alaninol residue instead of the allo-threonine.     

Sorting of newly synthesized galactosphingolipids to the two surface domains of epithelial cells

The high concentration of glycosphingolipids on the apical surface of epithelial cells may be generated by selective transport from their site of synthesis to the cell surface. Previously, we showed that canine kidney MDCK and human intestinal Caco-2 cells converted a ceramide carrying the short fluorescent fatty acid C6-NBD to glucosylceramide (GlcCer) and sphingomyelin (SM), and that GlcCer was preferentially transported to the apical surface as compared to SM. Here, we address the point that not all glycosphingolipid classes are apically enriched in epithelia. We show that a ceramide containing the 2-hydroxy fatty acid C6OH was preferentially converted by MDCK and Caco- 2 cells to galactosylceramide (GalCer) and its derivatives galabiosylceramide (Ga2Cer) and sulfatide (SGalCer) as compared to SM and GlcCer--all endogenous lipid classes of these cells. Transport to the apical and basolateral cell surface was monitored by a BSA- depletion assay. In MDCK cells, GalCer reached the cell surface with two- to sixfold lower apical/basolateral polarity than GlcCer. Remarkably, in Caco-2 cells GalCer and GlcCer displayed the same apical/basolateral polarity, but it was sixfold lower for lipids with a C6OH chain than for C6-NBD lipids. Therefore, the sorting of a sphingolipid appears to depend on lipid structure and cell type. We propose that the different ratios of gluco- and galactosphingolipid synthesis in the various epithelial tissues govern lipid sorting in the membrane of the trans Golgi network by dictating the composition of the domains from where vesicles bud to the apical and basolateral cell surface.     

Dissecting the mechanism and assembly of a complex virulence mycobacterial lipid

Mycobacterium tuberculosis cell envelope is a treasure house of biologically active lipids of fascinating molecular architecture. Although genetic studies have alluded to an array of genes in biosynthesis of complex lipids, their mechanistic, structural, and biochemical principles have not been investigated. Here, we have dissected the molecular logic underlying the biosynthesis of a virulence lipid phthiocerol dimycocerosate (PDIM). Cell-free reconstitution studies demonstrate that polyketide synthases, which are usually involved in the biosynthesis of secondary metabolites, are responsible for generating complex lipids in mycobacteria. We show that PapA5 protein directly transfers the protein bound mycocerosic acid analogs on phthiocerol to catalyze the final esterification step. Based on precise identification of biological functions of proteins from Pps cluster, we have rationally produced a nonmethylated variant of mycocerosate esters. Apart from elucidating mechanisms that generate chemical heterogeneity with PDIMs, this study also presents an attractive approach to explore host-pathogen interactions by altering mycobacterial surface coat.     

Accumulation of a GPI-anchored protein at the cell surface requires sorting at multiple intracellular levels

Proteins modified by glycosylphosphatidylinositol membrane anchors have become popular for investigating the role of membrane lipid microdomains in cellular sorting processes. To this end, trypanosomatids offer the advantage that they express these molecules in high abundance. The parasitic protozoan Trypanosoma brucei is covered by a dense and nearly homogeneous coat composed of a glycosylphosphatidylinositol-anchored protein, the variant surface glycoprotein, which is essential for survival of the parasite in the mammalian blood. Therefore, T. brucei must possess mechanisms to selectively and efficiently deliver variant surface glycoprotein to the cell surface. In this study, we have quantified the steady-state distribution of variant surface glycoprotein by differential biotinylation, by fluorescence microscopy and by immunoelectron microscopy on high-pressure frozen and freeze-substituted samples. These three techniques provide very similar estimates of the fraction of variant surface glycoprotein located on the cell surface, on average 89.4%. The intracellular variant surface glycoprotein (10.6%) is predominantly located in the endosomal compartment (75%), while 25% are associated with the endoplasmic reticulum, Golgi apparatus and lysosomes. The density of variant surface glycoprotein in the plasma membrane including the membrane of the flagellar pocket, the only site for endo- and exocytosis in this organism, is 48-52 times higher than the density in endoplasmic reticulum membranes. The relative densities of the Golgi complex and of the endosomes are 2.7 and 10.8, respectively, compared to the endoplasmic reticulum. This data set provides the basis for an analysis of the dynamics of sorting. Depending on the intracellular itinerary of newly formed variant surface glycoprotein, the high surface density is achieved in two (endoplasmic reticulum --> Golgi complex --> cell surface) or three enrichment steps (endoplasmic reticulum --> Golgi complex --> endosomes --> cell surface), suggesting sorting between several membrane compartments.     

Lipid transfer between endothelial and smooth muscle cells in coculture

A coculture system was employed to study the interactions between endothelium and vascular smooth muscle cells in arachidonic acid metabolism. Bovine aortic endothelial cells grown on micropore filters impregnated with gelatin and coated with fibronectin are mounted on polystyrene chambers and suspended over confluent smooth muscle cultures. The endothelial basal laminae are oriented toward the underlying smooth muscle, and the two layers are separated by only 1 mm. Each cell layer was assayed individually: apical and basolateral fluid also was collected separately for assay. Fatty acids, including arachidonic acid, are readily transferred between the endothelial and smooth muscle cells in this system. Distribution of the incorporated fatty acids among the lipids of each cell is the same as when the fatty acid is added directly to the culture medium. Arachidonic acid released from endothelial cells is available as a substrate for prostaglandin production by smooth muscle. In addition, fatty acids released from the smooth muscle cells can pass through the endothelium and accumulate in the fluid bathing the endothelial apical surface. These fatty acid interchanges may be involved in cell-cell signaling within the vascular wall, the clearance of lipids from the vascular wall, or the redistribution of arachidonic acid and other polyunsaturated fatty acids between adjacent cell types. Furthermore, the findings suggest that prostaglandin production by smooth muscle cells can occur in response to stimuli that cause arachidonic acid release from endothelial cells.     

The Na+/H+ Exchanger NHE6 in the Endosomal Recycling System Is Involved in the Development of Apical Bile Canalicular Surface Domains in HepG2 Cells

Polarized epithelial cells develop and maintain distinct apical and basolateral surface domains despite a continuous flux of membranes between these domains. The Na⁺/H⁺exchanger NHE6 localizes to endosomes but its function is unknown. Here, we demonstrate that polarized hepatoma HepG2 cells express an NHE6.1 variant that localizes to recycling endosomes and colocalizes with transcytosing bulk membrane lipids. NHE6.1 knockdown or overexpression decreases or increases recycling endosome pH, respectively, and inhibits the maintenance of apical, bile canalicular plasma membranes and, concomitantly, apical lumens. NHE6.1 knockdown or overexpression has little effect on the de novo biogenesis of apical surface domains. NHE6.1 knockdown does not inhibit basolateral-to-apical transcytosis of bulk membrane lipids, but it does promote their progressive loss from the apical surface, leaving cells unable to efficiently retain bulk membrane and bile canalicular proteins at the apical surface. The data suggest that a limited range of endosome pH mediated by NHE6.1 is important for securing the polarized distribution of membrane lipids at the apical surface and maintenance of apical bile canaliculi in HepG2 cells and hence cell polarity. This study underscores the emerging role of the endosomal recycling system in apical surface development and identifies NHE6 as a novel regulatory protein in this process.     

Identification and Recombinant Expression of a Mycobacterium avium Rhamnosyltransferase Gene (rtfA) Involved in Glycopeptidolipid Biosynthesis

The Mycobacterium avium complex is a source of disseminated infections in patients with advanced AIDS. This group of mycobacteria is distinguished by the presence of highly antigenic, surface-exposed glycopeptidolipids, and these glycolipids possess variant oligosaccharide structures that are the chemical basis of the 28 distinct serovars of the M. avium complex. We previously described the ser2 gene cluster, encoding the synthesis of the haptenic oligosaccharide (2,3-dimethylfucose-rhamnose-6-deoxytalose-) of the serovar 2-specific glycopeptidolipid, and revealed a locus (ser2A) encoding a putative rhamnosyltransferase. Sequencing of the ser2A locus demonstrated the presence of three open reading frames, two of which yielded significant homology to several glycosyltransferases, and the deduced amino acid sequences of these two putative glycosyltransferases had 63% identity. These two genes were expressed in Mycobacterium smegmatis, and the resulting recombinant glycopeptidolipids were characterized by thin-layer chromatography and gas chromatography-mass spectrometry. These analyses demonstrated that only one of these genes, termed rtfA, encoded the rhamnosyltransferase responsible for the transfer of rhamnose to 6-deoxytalose. The identification of rtfA will permit further evaluation of glycopeptidolipid biosynthesis and the construction of isogenic mutants of multiple M. avium complex serovars. Moreover, such mutants will help define the role of glycopeptidolipids in the intracellular survival of these bacteria.     

Temperature-sensitive transport of glycoproteins to the surface of a variant mouse lymphoma cell line.

The expression of mouse mammary tumor virus (MMTV) glycoproteins on the surface of stably infected mouse lymphoma cell line W7MG1 is dramatically increased by glucocorticoid hormones. A variant cell line, W7M.TS1, was selected from W7MG1 for its lack of expression of MMTV glycoproteins on the cell surface in response to treatment with glucocorticoid. Hormonal stimulation of MMTV RNA levels and hormone-induced cytolysis occurred normally in the variant cells. Furthermore, the rates of production of the precursor and mature forms of MMTV glycoproteins in the presence of glucocorticoid were similar in variant and wild-type cells. However, the accumulation of MMTV glycoproteins on the cell surface after hormone treatment was delayed by about 8 h in the variant relative to wild-type cells. The steady-state level of a constitutively expressed cellular protein, T200, on the variant cell surface was comparable to that on wild-type cells. However, in pulse-chase experiments, the appearance of newly synthesized T200 on the cell surface was delayed in the variant compared with wild-type cells. Another glucocorticoid hormone response, removal of H-2 class I antigens from the cell surface, was also delayed in the variant relative to wild-type cells, suggesting that turnover or internalization of cell surface glycoproteins may also be affected in the variant. The defects in the variant cell line were observed at 37 degrees C, but not at 31 degrees C; the variant cells grew normally at both temperatures. This variant phenotype defines a new genetic entity that is important for transport of glycoproteins between internal microsomal compartments and the cell surface.     

Mycobacterium abscessus glycopeptidolipid prevents respiratory epithelial TLR2 signaling as measured by HβD2 gene expression and IL-8 release

Mycobacterium abscessus has emerged as an important cause of lung infection, particularly in patients with bronchiectasis. Innate immune responses must be highly effective at preventing infection with M. abscessus because it is a ubiquitous environmental saprophyte and normal hosts are not commonly infected. M. abscessus exists as either a glycopeptidolipid (GPL) expressing variant (smooth phenotype) in which GPL masks underlying bioactive cell wall lipids, or as a variant lacking GPL which is immunostimulatory and invasive in macrophage infection models. Respiratory epithelium has been increasingly recognized as playing an important role in the innate immune response to pulmonary pathogens. Respiratory epithelial cells express toll-like receptors (TLRs) which mediate the innate immune response to pulmonary pathogens. Both interleukin-8 (IL-8) and human β-defensin 2 (HβD2) are expressed by respiratory epithelial cells in response to toll-like receptor 2 (TLR2) receptor stimulation. In this study, we demonstrate that respiratory epithelial cells respond to M. abscessus variants lacking GPL with expression of IL-8 and HβD2. Furthermore, we demonstrate that this interaction is mediated through TLR2. Conversely, M. abscessus expressing GPL does not stimulate expression of IL-8 or HβD2 by respiratory epithelial cells which is consistent with "masking" of underlying bioactive cell wall lipids by GPL. Because GPL-expressing smooth variants are the predominant phenotype existing in the environment, this provides an explanation whereby initial M. abscessus colonization of abnormal lung airways escapes detection by the innate immune system.     

Transmembrane and truncated (SEC) isoforms of MUC1 in the human endometrium and Fallopian tube

The cell surface mucin MUC1 is expressed by endometrial epithelial cells with increased abundance in the secretory phase of the menstrual cycle, when it is found both at the apical cell surface and in secretions. This suggests the presence of a maternal cell surface glycoprotein barrier to embryo implantation, arising from the anti-adhesive property of MUC1. In previous work, we demonstrated alternatively spliced MUC1 variant forms in tumour cells. The variant MUC1/SEC lacks the transmembrane and cytoplasmic sequences found in the full-length variant. We now show that MUC1/SEC mRNA is present in endometrial carcinoma cell lines, endometrial tissue and primary cultured endometrial epithelial cells. The protein can be detected using isoform-specific antibodies in uterine flushings, suggesting release from endometrium in vivo. However, on the basis of immunolocalisation studies, MUC1/SEC also remains associated with the apical epithelial surface both in tissue and in cultured cells. Transmembrane MUC1 and MUC1/SEC are both strikingly localised to the apical surface of tubal epithelium. Thus MUC1 may contribute to the anti-adhesive character of the tubal surface, inhibiting ectopic implantation. The mechanism by which this barrier is overcome in endometrium at implantation is the subject of ongoing investigation.     

The effect of a phorbol ester on the lipid microviscosity of two endoplasmic reticulum membrane fractions isolated from Krebs II ascites cells.

This paper deals with microviscosity parameters and thermoinduced structural transitions in the lipids of smooth and heavy rough endoplasmic reticulum membranes isolated from Krebs II ascites cells incubated with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate. The phorbol ester was found to bring about a threefold increase in the microviscosity of the lipids in heavy rough membranes. Spin probe I (2,2,6,6-tetrahydro-4-capryloyl-oxypiperidine-1-oxyl), localized in the surface layer of the membrane lipids, gave results which indicate an increased number of thermoinduced structural transitions in the smooth membranes in the treated cells due to the transitions occurring at relatively low temperature and a decreased number of such transitions in the heavy rough fraction especially at high temperature. For 5,6-benzo-2,2,4,4-tetramethyl-1,2,3,4-tetrahydro-gamma-carboline-oxyl, probe II, mainly distributed in the annular lipids, a decrease in the number of low temperature transitions in the smooth fraction was observed, while an increase occurred in the heavy rough one. The results obtained are discussed in terms of the effect of phorbol esters as promoters of tumor progression.