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Surface proteome of "Mycobacterium avium subsp. hominissuis" during the early stages of macrophage infection.

ABSTRACT: "Mycobacterium avium subsp. hominissuis" is a robust and pervasive environmental bacterium that can cause opportunistic infections in humans. The bacterium overcomes the host immune response and is capable of surviving and replicating within host macrophages. Little is known about the bacterial mechanisms that facilitate these processes, but it can be expected that surface-exposed proteins play an important role. In this study, the selective biotinylation of surface-exposed proteins, streptavidin affinity purification, and shotgun mass spectrometry were used to characterize the surface-exposed proteome of M. avium subsp. hominissuis. This analysis detected more than 100 proteins exposed at the bacterial surface of M. avium subsp. hominissuis. Comparisons of surface-exposed proteins between conditions simulating early infection identified several groups of proteins whose presence on the bacterial surface was either constitutive or appeared to be unique to specific culture conditions. This proteomic profile facilitates an improved understanding of M. avium subsp. hominissuis and how it establishes infection. Additionally, surface-exposed proteins are excellent targets for the host adaptive immune system, and their identification can inform the development of novel treatments, diagnostic tools, and vaccines for mycobacterial disease.

SUBMITTER: McNamara M

PROVIDER: S-EPMC3347459 | biostudies-other | 2012 May

REPOSITORIES: biostudies-other

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Surface proteome of "Mycobacterium avium subsp. hominissuis" during the early stages of macrophage infection.

McNamara Michael M Tzeng Shin-Cheng SC Maier Claudia C Zhang Li L Bermudez Luiz E LE

Infection and immunity 20120305 5

"Mycobacterium avium subsp. hominissuis" is a robust and pervasive environmental bacterium that can cause opportunistic infections in humans. The bacterium overcomes the host immune response and is capable of surviving and replicating within host macrophages. Little is known about the bacterial mechanisms that facilitate these processes, but it can be expected that surface-exposed proteins play an important role. In this study, the selective biotinylation of surface-exposed proteins, streptavidi ...[more]

PMID: 22392927

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Project description:Johne's disease is a chronic and usually fatal enteric infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP) and is responsible for hundreds of millions of dollars in losses for the agricultural industry. Natural infection typically begins with bacterial uptake and translocation through the epithelium of the small intestine, followed by ingestion by tissue macrophages and dissemination via the lymphatic or blood system throughout the body. To gain insights into the host responses and adaptation of MAP within phagocytic cells, we utilized the previously developed cell culture passage model, and mass spectrometric-based quantitative proteomic approach. Using the cell culture system, which mimics an in vivo interaction of MAP with intestinal epithelium and tissue macrophages, bacteria were passed through the bovine epithelial cells and, subsequently, used for macrophage infection (termed indirect infection), while uninfected cells and macrophage infection initiated with the culture grown bacteria (termed direct infection) served as controls. Approximately 3900 proteins were identified across all studied groups. The comparison within the subset of proteins that showed synthesis for more than two-fold in the direct infection over the uninfected control revealed an enrichment for the pro-inflammatory pathways such as the NF-κB and cytokine/chemokine signaling, positive regulation of defense response, cell activation involved in the immune response and adaptive immune system. While these responses were absent in the indirect infection, cellular pathways such as cell cycle, healing, regulation of cell adhesion, ensemble of core extracellular matrix proteins, cell surface integrins and proteins mediating the integrin signaling were remarkably high within the indirect infection. In addition to global analysis of the macrophage proteome, we further validated the proteomics data and confirmed that MAP passage through epithelial cells modulates the expression and signaling of integrins in phagocytes. In this study, we demonstrate that predominant expression of integrins in the indirectly infected macrophages allows phagocytic cells to initiate stronger binding and efficient translocation through the endothelial cells, suggesting the important role of integrins in the spread of MAP infection.

Project description:BackgroundMycobacterium avium subspecies avium (M. avium) is frequently encountered in the environment, but also causes infections in animals and immunocompromised patients. In contrast, Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a slow-growing organism that is the causative agent of Johne's disease in cattle and chronic granulomatous infections in a variety of other ruminant hosts. Yet we show that despite their divergent phenotypes and the diseases they present, the genomes of M. avium and M. paratuberculosis share greater than 97% nucleotide identity over large (25 kb) genomic regions analyzed in this study.ResultsTo characterize genome similarity between these two subspecies as well as attempt to understand their different growth rates, we designed oligonucleotide primers from M. avium sequence to amplify 15 minimally overlapping fragments of M. paratuberculosis genomic DNA encompassing the chromosomal origin of replication. These strategies resulted in the successful amplification and sequencing of a contiguous 11-kb fragment containing the putative Mycobacterium paratuberculosis origin of replication (oriC). This fragment contained 11 predicted open reading frames that showed a conserved gene order in the oriC locus when compared with several other Gram-positive bacteria. In addition, a GC skew analysis identified the origin of chromosomal replication which lies between the genes dnaA and dnaN. The presence of multiple DnaA boxes and the ATP-binding site in dnaA were also found in M. paratuberculosis. The strong nucleotide identity of M. avium and M. paratuberculosis in the region surrounding the origin of chromosomal replication led us to compare other areas of these genomes. A DNA homology matrix of 2 million nucleotides from each genome revealed strong synteny with only a few sequences present in one genome but absent in the other. Finally, the 16s rRNA gene from these two subspecies is 100% identical.ConclusionsWe present for the first time, a description of the oriC region in M. paratuberculosis. In addition, genomic comparisons between these two mycobacterial subspecies suggest that differences in the oriC region may not be significant enough to account for the diverse bacterial replication rates. Finally, the few genetic differences present outside the origin of chromosomal replication in each genome may be responsible for the diverse growth rates or phenotypes observed between the avium and paratuberculosis subspecies.

Project description:The genetic similarity between Mycobacterium avium subsp. paratuberculosis and other mycobacterial species has confounded the development of M. avium subsp. paratuberculosis-specific diagnostic reagents. Random shotgun sequencing of the M. avium subsp. paratuberculosis genome in our laboratories has shown >98% sequence identity with Mycobacterium avium subsp. avium in some regions. However, an in silico comparison of the largest annotated M. avium subsp. paratuberculosis contigs, totaling 2,658,271 bp, with the unfinished M. avium subsp. avium genome has revealed 27 predicted M. avium subsp. paratuberculosis coding sequences that do not align with M. avium subsp. avium sequences. BLASTP analysis of the 27 predicted coding sequences (genes) shows that 24 do not match sequences in public sequence databases, such as GenBank. These novel sequences were examined by PCR amplification with genomic DNA from eight mycobacterial species and ten independent isolates of M. avium subsp. paratuberculosis. From these analyses, 21 genes were found to be present in all M. avium subsp. paratuberculosis isolates and absent from all other mycobacterial species tested. One region of the M. avium subsp. paratuberculosis genome contains a cluster of eight genes, arranged in tandem, that is absent in other mycobacterial species. This region spans 4.4 kb and is separated from other predicted coding regions by 1,408 bp upstream and 1,092 bp downstream. The gene upstream of this eight-gene cluster has strong similarity to mycobacteriophage integrase sequences. The GC content of this 4.4-kb region is 66%, which is similar to the rest of the genome, indicating that this region was not horizontally acquired recently. Southern hybridization analysis confirmed that this gene cluster is present only in M. avium subsp. paratuberculosis. Collectively, these studies suggest that a genomics approach will help in identifying novel M. avium subsp. paratuberculosis genes as candidate diagnostic sequences.

Dataset Information

Surface proteome of "Mycobacterium avium subsp. hominissuis" during the early stages of macrophage infection.

Publications

Surface proteome of "Mycobacterium avium subsp. hominissuis" during the early stages of macrophage infection.

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