Torsten M. Eckstein

A genetic mechanism for deletion of the ser2 gene cluster and formation of rough morphological variants of Mycobacterium avium.

A major phenotypic trait of the Mycobacterium avium complex is the ability to produce rough and smooth colony variants. The chemical basis of this morphological variation is the loss of an antigenic surface structure, termed glycopeptidolipid (GPL), by rough variants. Using M. avium serovar 2 strain 2151 as a model system, this laboratory previously reported that rough variants arise via the deletion of large genomic regions encoding GPL biosynthesis. One such deletion encompasses the gene cluster (ser2) responsible for production of the serovar 2 GPL haptenic oligosaccharide. In this study, nucleotide sequencing revealed that both ends of the ser2 gene cluster are flanked by a novel insertion sequence (IS1601) oriented as direct repeats. Detailed analyses of the site of deletion in the genome of M. avium 2151 Rg-1 demonstrated that a single copy of IS1601 remained and that the ser2 gene cluster was deleted by homologous recombination. This same deletion pattern was observed for 10 out of 15 rough colony variants tested. Additionally, these studies revealed that IS1601 contains portions of three independent insertion sequences. This report is the first to define the precise genetic basis of colony variation in Mycobacterium spp. and provides further evidence that homologous recombination between insertion sequence elements can be a primary determinant of genome plasticity in these bacteria.

A novel cell wall lipopeptide is important for biofilm formation and pathogenicity of Mycobacterium avium subspecies paratuberculosis.

Biofilm formation by pathogenic bacteria plays a key role in their pathogenesis. Previously, the pstA gene was shown to be involved in the virulence of Mycobacterium avium subspecies paratuberculosis (M. ap), the causative agent of Johnes disease in cattle and a potential risk factor for Crohns disease. Scanning electron microscopy and colonization levels of the M. ap mutant indicated that the pstA gene significantly contributes to the ability of M. ap to form biofilms. Digital measurements taken during electron microscopy identified a unique morphology for the DeltapstA mutant, which consisted of significantly shorter bacilli than the wild type. Analysis of the lipid profiles of the mycobacterial strains identified a novel lipopeptide that was present in the cell wall extracts of wild-type M. ap, but missing from the DeltapstA mutant. Interestingly, the calf infection model suggested that pstA contributes to intestinal invasion of M. ap. Furthermore, immunoblot analysis of peptides encoded by pstA identified a specific and significant level of immunogenicity. Taken together, our analysis revealed a novel cell wall component that could contribute to biofilm formation and to the virulence and immunogenicity of M. ap. Molecular tools to better control M. ap infections could be developed utilizing the presented findings.

A Major Cell Wall Lipopeptide of Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne disease in cattle and other ruminants, is proposed to be at least one of the causes of Crohn disease in humans. MAP and Mycobacterium avium subspecies avium, a closely related opportunistic environmental bacterium, share 95% of their genes and exhibit homologies of more than 99% between these genes. The identification of molecules specific for MAP is essential for understanding its pathogenicity and for development of useful diagnostic tools. The application of gas chromatography, mass spectrometry, and nuclear magnetic resonance led to the structural identification of a major cell wall lipopeptide of MAP, termed Para-LP-01, defined as C20 fatty acyl-d-Phe-N-Me-l-Val-l-Ile-l-Phe-l-Ala methyl ester. Variations of this lipopeptide with different fatty acyl moieties (C16 fatty acyl through C17, C18, C19, C21 to C22) were also identified. Besides the specificity of this lipopeptide for MAP, the presence of an N-Me-l-valine represents the first reported N-methylated amino acid within an immunogenic lipopeptide of mycobacteria. Sera from animals with Johne disease, but not sera from uninfected cattle, reacted with this lipopeptide, indicating potential biological importance.

A Mycobacterium avium subsp. paratuberculosis LuxR regulates cell envelope and virulence.

Mycobacterium avium subsp. paratuberculosis adapts to the environment via the regulation of genes affecting its envelope’s composition. Bacteria grown in milk (in vivo conditions) presented differences in the cell wall-associated lipids and in the expression of genes involved in lipid metabolism (FadE8, FadE6 and MAP1420) and host cell invasion (MAP1203, LprL). A different lipid profile was also observed in the envelope of intracellular bacteria after 1 h of infection. Intracellular bacteria showed up-regulation of a LuxR regulator which controls the envelope’s composition by up-regulation of FadE8, MAP1420, MAP1203 and LprL and by down-regulation of pks12, mmpL2 and MAP2594. A LuxR-overexpressing strain with a lipid-deficient envelope phenotype, infected epithelial cells more efficiently than the wild-type bacteria; however, it was not more resistant than the wild-type strain to the action of bactericidal proteins. Here we show that LuxR regulates virulence determinants and is involved in mycobacteria adaptation to the host.

Mannosylated lipoarabinomannans from Mycobacterium avium subsp. paratuberculosis alters the inflammatory response by bovine macrophages and suppresses killing of Mycobacterium avium subsp. avium organisms.

Analysis of the mechanisms through which pathogenic mycobacteria interfere with macrophage activation and phagosome maturation have shown that engagement of specific membrane receptors with bacterial ligands is the initiating event. Mannosylated lipoarabinomannan (Man-LAM) has been identified as one of the ligands that modulates macrophage function. We evaluated the effects of Man-LAM derived from Mycobacterium avium subsp. paratuberculosis (MAP) on bovine macrophages. Man-LAM induced a rapid and prolonged expression of IL-10 message as well as transient expression of TNF-α. Preincubation with Man-LAM for up to 16 h did not suppress expression of IL-12 in response to interferon-γ. Evaluation of the effect of Man-LAM on phagosome acidification, phagosome maturation, and killing of Mycobacterium avium subsp. avium (MAA) showed that preincubation of macrophages with Man-LAM before addition of MAA inhibited phagosome acidification, phagolysosome fusion, and reduced killing. Analysis of signaling pathways provided indirect evidence that inhibition of killing was associated with activation of the MAPK-p38 signaling pathway but not the pathway involved in regulation of expression of IL-10. These results support the hypothesis that MAP Man-LAM is one of the virulence factors facilitating survival of MAP in macrophages.

Reduced transcript stabilization restricts TNF-α expression in RAW264.7 macrophages infected with pathogenic mycobacteria: evidence for an involvement of lipomannan

Despite the critical role that TNF‐α plays in the containment of mycobacterial infection, the mechanisms involved in regulation of its expression by mycobacteria are poorly defined. We addressed this question by studying MAP, which causes a chronic enteritis in ruminants and is linked to human Crohns disease. We found that in MAP‐infected macrophages, TNF‐α gene expression was substantially lower than in macrophages infected with nonpathogenic MS or stimulated with LPS. TNF‐α transcription alone could not fully explain the differential TNF‐α mRNA expression, suggesting that there must be a substantial contribution by post‐transcriptional mechanisms. Accordingly, we found reduced TNF‐α mRNA stability in MAP‐infected macrophages. Further comparison of MAP‐ and MS‐infected macrophages revealed that lower TNF‐α mRNA stability combined with lower mRNA and protein expression in MAP‐infected macrophages correlated with lower p38 MAPK phosphorylation. Theses findings were independent of viability of MAP and MS. We demonstrate that the major mycobacterial cell‐wall lipoglycan LM of MAP and MS induced TNF‐α mRNA transcription, but only the MS‐LM induced p38 MAPK‐dependent transcript stabilization. Overall, our data suggest that pathogenic mycobacteria cause weak p38 MAPK activation and TNF‐α mRNA stabilization as a result of their structural cell‐wall components such as LM and thereby, restrict TNF‐α expression in macrophages.

Infection with Mycobacterium avium subsp. paratuberculosis Results in Rapid Interleukin-1β Release and Macrophage Transepithelial Migration

ABSTRACT Pathogen processing by the intestinal epithelium involves a dynamic innate immune response initiated by pathogen-epithelial cell cross talk. Interactions between epithelium and Mycobacterium avium subsp. paratuberculosis have not been intensively studied, and it is currently unknown how the bacterium-epithelial cell cross talk contributes to the course of infection. We hypothesized that M. avium subsp. paratuberculosis harnesses host responses to recruit macrophages to the site of infection to ensure its survival and dissemination. We investigated macrophage recruitment in response to M. avium subsp. paratuberculosis using a MAC-T bovine macrophage coculture system. We show that M. avium subsp. paratuberculosis infection led to phagosome acidification within bovine epithelial (MAC-T) cells as early as 10 min, which resulted in upregulation of interleukin-1β (IL-1β) at transcript and protein levels. Within 10 min of infection, macrophages were recruited to the apical side of MAC-T cells. Inhibition of phagosome acidification or IL-1β abrogated this response, while MCP-1/CCL-2 blocking had no effect. IL-1β processing was dependent upon Ca2+ uptake from the extracellular medium and intracellular Ca2+ oscillations, as determined by EGTA and BAPTA-AM [1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester)] treatments. Thus, M. avium subsp. paratuberculosis is an opportunist that takes advantage of extracellular Ca2+-dependent phagosome acidification and IL-1β processing in order to efficiently transverse the epithelium and enter its niche—the macrophage.

Utilization of a ts-sacB selection system for the generation of a Mycobacterium avium serovar-8 specific glycopeptidolipid allelic exchange mutant

BackgroundMycobacterium avium are ubiquitous environmental organisms and a cause of disseminated infection in patients with end-stage AIDS. The glycopeptidolipids (GPL) of M. avium are proposed to participate in the pathogenesis of this organism, however, establishment of a clear role for GPL in disease production has been limited by the inability to genetically manipulate M. avium.MethodsTo be able to study the role of the GPL in M. avium pathogenesis, a ts-sacB selection system, not previously used in M. avium, was employed as a means to achieve homologous recombination for the rhamnosyltransferase (rtfA) gene of a pathogenic serovar 8 strain of M. avium to prevent addition of serovar-specific sugars to rhamnose of the fatty acyl-peptide backbone of GPL. The genotype of the resultant rtfA mutant was confirmed by polymerase chain reaction and southern hybridization. Disruption in the proximal sugar of the haptenic oligosaccharide resulted in the loss of serovar specific GPL with no change in the pattern of non-serovar specific GPL moieties as shown by thin layer chromatography and gas chromatography/mass spectrometry. Complementation of wild type (wt) rtfA in trans through an integrative plasmid restored serovar-8 specific GPL expression identical to wt serovar 8 parent strain.ResultsIn this study, we affirm our results that rtfA encodes an enzyme responsible for the transfer of Rha to 6d-Tal and provide evidence of a second allelic exchange mutagenesis system suitable for M. avium.ConclusionWe report the second allelic exchange system for M. avium utilizing ts-sacB as double-negative and xylE as positive counter-selection markers, respectively. This system of allelic exchange would be especially useful for M. avium strains that demonstrate significant isoniazid (INH) resistance despite transformation with katG. Through the construction of mutants in GPL or other mycobacterial components, their roles in M. avium pathogenesis, biosynthesis, or drug resistance can be studied in a consistent manner.

Cellular and humoral immunogenicity of Mycobacterium avium subsp. paratuberculosis specific lipopentapeptide antigens

Paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (MAP) is a chronic infectious disease affecting domestic and wild ruminants. Antigens currently used for the diagnosis of paratuberculosis are whole-cell derived crude preparations. The identification of MAP-specific antigens for the specific and early diagnosis of this infection is strongly needed. This study assessed the ability of the MAP-specific synthetic lipopeptide antigen Para-LP-01 to invoke specific serum antibody (Ab) and cell-mediated immune (CMI) responses in sheep experimentally exposed to MAP S strain. Responses were compared to those elicited by the crude whole-cell derived MAP 316v antigen (316v). Para-LP-01 induced a significant serum Ab response in MAP-infected sheep in comparison with unexposed or uninfected sheep, but failed to induce detectable CMI responses including production of IFN-γ, IL-10 and lymphoproliferation, unlike 316v which invoked both CMI and serum Ab responses in MAP-exposed sheep. Para-LP-01 is a suitable antigen for serodiagnosis of MAP-infection in sheep. The differential induction of humoral and CMI responses by lipid based antigens could enhance current understanding of the role played by cell-wall associated lipid antigens in the pathogenesis of MAP-infection.

Characterization of the inflammatory phenotype of Mycobacterium avium subspecies paratuberculosis using a novel cell culture passage model

Understanding the pathogenic mechanisms of Mycobacterium avium subspecies paratuberculosis (MAP) and the host responses to Johnes disease is complicated by the multi-faceted disease progression, late-onset host reaction and the lack of available ex vivo infection models. We describe a novel cell culture passage model that mimics the course of infection in vivo. The developed model simulates the interaction of MAP with the intestinal epithelial cells, followed by infection of macrophages and return to the intestinal epithelium. MAP internalization triggers a minimal inflammatory response. After passage through a macrophage phase, bacterial reinfection of MDBK epithelial cells, representing the late phase of intestinal mucosal infection, is associated with increased synthesis of the pro-inflammatory transcripts of IL-6, CCL5, IL-8 and IL-18, paired with decreased levels of TGFβ. Transcriptome analysis of MAP from each stage of epithelial cell infection identified increased expression of lipid biosynthesis and lipopeptide modification genes in the inflammatory phenotype of MAP. Total lipid analysis by HPLC-ES/MS indicates different lipidomic profiles between the two phenotypes and a unique set of lipids composing the inflammatory MAP phenotype. The presence of selected upregulated lipid-modification gene transcripts in samples of ileal tissue from cows diagnosed with Johnes disease supports and validates the model. By using the relatively simple cell culture passage model, we show that MAP alters its lipid composition during intracellular infection and acquires a pro-inflammatory phenotype, which likely is associated with the inflammatory phase of Johnes disease.