Frederick D. Quinn

Necrosis of Lung Epithelial Cells during Infection with Mycobacterium tuberculosis Is Preceded by Cell Permeation

ABSTRACT Mycobacterium tuberculosis establishes infection, progresses towards disease, and is transmitted from the alveolus of the lung. However, the role of the alveolar epithelium in any of these pathogenic processes of tuberculosis is unclear. In this study, lung epithelial cells (A549) were used as a model in which to examine cytotoxicity during infection with either virulent or avirulent mycobacteria in order to further establish the role of the lung epithelium during tuberculosis. Infection of A549 cells with M. tuberculosis strains Erdman and CDC1551 demonstrated significant cell monolayer clearing, whereas infection with eitherMycobacterium bovis BCG or Mycobacterium smegmatis LR222 did not. Clearing of M. tuberculosis-infected A549 cells correlated to necrosis, not apoptosis. Treatment of M. tuberculosis-infected A549 cells with streptomycin, but not cycloheximide, demonstrated a significant reduction in the necrosis of A549 cell monolayers. This mycobacterium-induced A549 necrosis did not correlate to higher levels of intracellular or extracellular growth by the mycobacteria during infection. Staining of infected cells with propidium iodide demonstrated that M. tuberculosis induced increased permeation of A549 cell membranes within 24 h postinfection. Quantitation of lactate dehydrogenase (LDH) release from infected cells further demonstrated that cell permeation was specific to M. tuberculosis infection and correlated to A549 cellular necrosis. Inactivated M. tuberculosis or its subcellular fractions did not result in A549 necrosis or LDH release. These studies demonstrate that lung epithelial cell cytotoxicity is specific to infection by virulent mycobacteria and is caused by cellular necrosis. This necrosis is not a direct correlate of mycobacterial growth or of the expression of host cell factors, but is preceded by permeation of the A549 cell membrane and requires infection with live bacilli.

Characterization of the Twin-Arginine Translocase Secretion System of Mycobacterium smegmatis

The twin-arginine translocation (TAT) system secretes fully folded proteins that contain a twin-arginine motif within their signal sequence across the cytoplasmic membrane in bacteria. Using a green fluorescent protein fused with a TAT signal sequence, we demonstrated that Mycobacterium smegmatis contains a TAT system. By inactivating individual genes, we showed that three genes (tatA, tatB, and tatC) are required for a functional TAT system in M. smegmatis. The tat mutants exhibited a decreased growth rate and altered colony morphology compared to the parent strain. Comparison of the secreted proteins of the deltatatC and parent strain by two-dimensional polyacrylamide gel electrophoresis revealed an alteration in the secretion of at least five proteins, and one of the major TAT-dependent secreted proteins was identified as beta-lactamase (BlaS). The genome of M. smegmatis was analyzed with the TATFIND program, and 49 putative TAT substrates were identified, including the succinate transporter DctP. Because disruption of the TAT secretion system has a direct effect on the physiology of M. smegmatis and homologs of the TAT proteins are also present in the genome of Mycobacterium tuberculosis, the TAT secretion system or its substrates may be good candidates for drug or vaccine development.

An in vitro model of the leukocyte interactions associated with granuloma formation in Mycobacterium tuberculosis infection

The principal defense of the human host against a Mycobacterium tuberculosis infection is the formation of granulomas, organized collections of activated macrophages, including epithelioid and multinucleated giant cells, surrounded by lymphocytes. This granuloma can sequester and contain the bacteria preventing active disease, and if the granuloma is maintained, these bacteria may remain latent for a persons lifetime. Secretion of a variety of chemoattractant cytokines following phagocytosis of the bacilli by the macrophage is critical not only to the formation of the granuloma but also to its maintenance. To investigate this process of early granuloma formation, we developed an in vitro model composed entirely of human cells. Combining blood lymphocytes and autologous macrophages from healthy purified protein derivative skin test‐negative individuals and mycobacteria resulted in the formation of small, rounded aggregate structures. Microscopic examination found macrophage‐specific CD68+ epithelioid macrophages and small round CD3+ lymphocytes that in complex resembled small granulomas seen in clinical pathology specimens. Acid‐fast staining bacteria were observed between and possibly within the cells composing the granulomas. Supernatants from the infected cells collected at 24 and 48 h and 5 and 9 days after infection were analyzed by a multiplexed cytokine bead‐based assay using the Luminex 100 and were found to contain interleukin (IL)‐6, IL‐8, interferon‐γ and tumor necrosis factor‐α, cytokines known to be involved in human granuloma formation, in quantities from two‐fold to 7000‐fold higher than supernatants from uninfected control cells. In addition, chemotaxis assays demonstrated that the same supernatants attracted significantly more human peripheral blood mononuclear cells than those of uninfected cells (P<0.001). This model may provide insight into the earliest stages of granuloma formation in those newly infected.

Involvement of Matrix Metalloproteinases in Human Immunodeficiency Virus Type 1-Induced Replication by Clinical Mycobacterium avium Isolates

The role of Mycobacterium avium isolates in modulating human immunodeficiency virus type 1 (HIV-1) replication was examined by use of an in vitro, resting T cell system. Two human clinical isolates (serotypes 1 and 4) but not an environmental M. avium isolate (serotype 2) enhanced HIV-1 replication. The M. avium-induced HIV-1 replication was not associated with cell activation or differential cytokine production or utilization. Addition of matrix metalloproteinase (MMP) inhibitors and their in vivo regulators, tissue inhibitors of metalloproteinases-1 and -2, abrogated M. avium-induced HIV-1 replication 80%-95%. The MMP inhibitors did not have any effect on the HIV-1 protease activity, suggesting that they may affect cellular processes. Furthermore, MMP-9 protein was differentially expressed after infection with clinical M. avium isolates and paralleled HIV-1 p24 production. Collectively, these data suggest that M. avium-induced HIV-1 replication is mediated, in part, through the induction of MMP-9.

Increased expression of Borrelia burgdorferi vlsE in response to human endothelial cell membranes

RNA isolated from virulent Borrelia burgdorferi cells incubated with human endothelial or neurological tissue cells was subjected to subtractive hybridization using RNA from the same strain incubated in tissue culture medium alone. This RNA subtractive technique generated specific probes that hybridized to two restriction fragments (8.2 kb and 10 kb respectively) generated by EcoRI digestion of total plasmid DNA. The 10 kb EcoRI fragment localized to lp28‐1 and was subsequently identified as the variable membrane protein‐like sequence (vls) region, which includes an expression locus (vlsE) and 15 silent cassettes. vlsE encodes a 36 kDa outer surface protein that undergoes antigenic variation during animal infections. Primer extension analysis identified the 5′ end of a transcript and a putative promoter for vlsE. Quantitative reverse transcription–polymerase chain reaction (RT–PCR) suggested that the expression of vlsE increased when virulent B. burgdorferi cells were incubated with human tissue cells or purified cell membranes isolated from those same cell lines. A 138 bp region upstream of the vlsE region that was not reported in the genome sequence was sequenced using specific 32P end‐labelled primers in a DNA cycle sequencing system at high annealing temperatures. Analysis revealed that it contained a 51 bp inverted repeat, which could form an extremely stable cruciform structure. Southern blots probed with the vlsE promoter/operator region indicated that part or all of this sequence could be found on other B. burgdorferi plasmids.

Influence of Mycobacterium avium subsp. paratuberculosis on Colitis Development and Specific Immune Responses during Disease

ABSTRACT The granulomatous and intramural inflammation observed in cases of inflammatory bowel diseases (IBD) and veterinary Johnes disease suggests that Mycobacterium avium subsp. paratuberculosis is a causative agent. However, an incomplete understanding of the immunological steps responsible for the pathologies of IBD makes this conclusion uncertain. Sera from interleukin-10-deficient (IL-10−/−) mice with spontaneous colitis displayed significantly higher M. avium subsp. paratuberculosis-specific immunoglobulin G2a antibody responses than did sera from similar mice without disease. Pathogen-free IL-10−/− mice received control vehicle or the vehicle containing heat-killed or live M. avium subsp. paratuberculosis. Mucosal CD4+ T cells from the mice that developed colitis proliferated and secreted higher levels of gamma interferon and tumor necrosis factor alpha after ex vivo stimulation with a Vβ11+ T-cell receptor-restricted peptide from the MPT59 antigen (Ag85B) than those secreted from cells from mice before the onset of colitis. The data from this study provide important information regarding the mechanisms of colitis in IL-10−/− mice, which are driven in part by Ag85B-specific T cells. The data suggest a plausible mechanism of Ag-specific T-cell responses in colitis driven by potent Ags conserved in Mycobacterium species.

Involvement of the autophagy pathway in trafficking of Mycobacterium tuberculosis bacilli through cultured human type II epithelial cells

Interactions between Mycobacterium tuberculosis bacilli and alveolar macrophages have been extensively characterized, while similar analyses in epithelial cells have not been performed. In this study, we microscopically examined endosomal trafficking of M. tuberculosis strain Erdman in A549 cells, a human type II pneumocyte cell line. Immuno‐electron microscopic (IEM) analyses indicate that M. tuberculosis bacilli are internalized to a compartment labelled first with Rab5 and then with Rab7 small GTPase proteins. This suggests that, unlike macrophages, M. tuberculosis bacilli traffic to late endosomes in epithelial cells. However, fusion of lysosomes with the bacteria‐containing compartment appears to be inhibited, as illustrated by IEM studies employing LAMP‐2 and cathepsin‐L antibodies. Examination by transmission electron microscopy and IEM revealed M. tuberculosis‐containing compartments surrounded by double membranes and labelled with antibodies against the autophagy marker Lc3, providing evidence for involvement and intersection of the autophagy and endosomal pathways. Interestingly, inhibition of the autophagy pathway using 3‐methyladenine improved host cell viability and decreased numbers of viable intracellular bacteria recovered after 72 h post infection. Collectively, these datasuggest that trafficking patterns for M. tuberculosis bacilli in alveolar epithelial cells differ from macrophages, and that autophagy is involved this process.

In search of virulence factors of human bacterial disease

Traditional genetic techniques and a variety of animal and tissue-culture model systems have sustained the study of bacterial virulence mechanisms for several decades. However, the recent application of newly developed molecular and cellular techniques has brought our understanding of bacterial pathogenesis to new heights by permitting the identification and analysis of previously unknown constitutively and differentially expressed virulence-associated factors.

Identification of mycobacteria based on spectroscopic analyses of mycolic acid profiles

This report examines lipophilic extracts containing mycolic acids isolated from tuberculosis (MTB) and non-tuberculosis (NTM) mycobacterial strains using chromatography, mass spectrometry (MS), nuclear magnetic resonance (NMR), and Raman spectroscopy. Gas chromatography-MS was used to identify major fatty acid mycolate components, while proton NMR confirmed the presence of characteristic cis- and trans-cyclopropane rings within different mycolic acid sub-types. Surface-enhanced Raman (SERS) spectra were obtained from the mycolic acids extracted from the bacterial cell envelopes of the MTB or NTM mycobacterial species. The Raman spectral profiles were used to develop a classification method based on chemometrics for identification of the mycobacterial species. Multivariate statistical analysis methods, including principal component analysis (PCA), hierarchical cluster analysis (HCA), and partial least squares discriminant analysis (PLS-DA) of the SERS spectra enabled differentiation of NTM mycobacteria from one another with 100% accuracy. These methods are also sensitive enough to differentiate clinically-isolated MTB strains that differed only by the presence or absence of a single extracytoplasmic sigma factor with 83-100% sensitivity and 80-100% specificity. The current work is the first report on discrimination of mycobacteria strains based on the SERS spectra of the constituent mycolic acids in lipophilic extracts. These results suggest that SERS can be used as an accurate and sensitive method for species and strain discrimination in mycobacteria.

CXCL10+ T cells and NK cells assist in the recruitment and activation of CXCR3+ and CXCL11+ leukocytes during Mycobacteria-enhanced colitis.

BackgroundThe role of Mycobacteria in the etiology of Crohns disease (CD) has been a contentious subject for many years. Recently, our laboratory showed that spontaneous colitis in IL-10-/- mice is driven in part by antigens (Ags) conserved in Mycobacteria. The present study dissects some of the common cellular and molecular mechanism that drive Mycobacteria-mediated and spontaneous colitis in IL-10-/- mice.ResultsWe show that serum from inflammatory bowel disease (IBD) patients contain significantly higher levels of Mycobacterium avium paratuberculosis-specific IgG1 and IgG2 antibodies (Abs), serum amyloid A (SAA) as well as CXCR3 ligands than serum from healthy donors. To study the cellular mechanisms of Mycobacteria-associated colitis, pathogen-free IL-10-/- mice were given heat-killed or live M. avium paratuberculosis. The numbers of mucosal T cells, neutrophils, NK/NKT cells that expressed TNFα, IFN-γ, and/or CXCL10 were significantly higher in mice that received live Mycobacteria than other groups. The numbers of mucosal CXCR3+, CXCL9+, CXCL11+ and/or IFN-γ+ dendritic cells (DCs) were also significantly higher in M. avium paratuberculosis-challenged mice, than compared to control mice.ConclusionThe present study shows that CD and UC patients mount significant Mycobacteria-specific IgG1 > IgG2 and CXCR3 ligand responses. Several cellular mechanisms that drive spontaneous colitis also mediate Mycobacteria-enhanced colitis in IL-10-/- mice. Similar to IL-10-/- mice under conventional housing, we show that Mycobacteria-challenge IL-10-/- mice housed under otherwise pathogen-free conditions develop colitis that is driven by CXCR3- and CXCR3 ligand-expressing leukocytes, which underscores another important hallmark and molecular mechanism of colitis. Together, the data show that Mycobacteria-dependent host responses, namely CXCL10+ T cells and NK cells, assist in the recruitment and activation of CXCR3+ and CXCL11+ leukocytes to enhance colitis of susceptible hosts.