No Holes Barred: Invasion of the Intestinal Mucosa by Mycobacterium avium subsp. paratuberculosis

The infection biology of Mycobacterium avium subsp. paratuberculosis has recently crystallized, with added details surrounding intestinal invasion. The involvement of pathogen-derived effector proteins such as the major membrane protein, oxidoreductase, and fibronectin attachment proteins have been uncovered. Mutations constructed in this pathogen have also shed light on genes needed for invasion. The host cell types that are susceptible to invasion have been defined, along with their transcriptional response. Recent details have given a new appreciation for the dynamic interplay between the host and bacterium that occurs at the outset of infection. An initial look at the global expression pathways of the host has shown a circumvention of the cell communication pathway by M. avium subsp. paratuberculosis, which loosens the integrity of the tight junctions. We now know that M. avium subsp. paratuberculosis activates the epithelial layer and also actively recruits macrophages to the site of infection. These notable findings are summarized along with added mechanistic details of the early infection model. We conclude by pro- posing critical next steps to further elucidate the process of M. avium subsp. paratuberculosis invasion

Mycobacterium avium resists exposure to the acidic conditions of the stomach

Organisms of the Mycobacterium avium complex are common pathogens in immunosuppressed patients such as individuals with AIDS. There is evidence that in AIDS patients, the main route for M. avium infection is the gastrointestinal tract. The stomach is a formidable barrier to pathogens and the ability to resist exposure to pH lower than 3 has been shown to be a virulence determinant of enteric pathogens. Incubation of three clinical isolates of M. avium under acidic pH revealed resistance of M. avium grown both to the exponential and stationary phase at pH 2.2 for 2 h. Inhibition of protein synthesis had no effect on the acid tolerance. When the duration of the incubation at pH 2.2 was extended to 24 h, bacteria grown to the stationary phase had a significantly greater tolerance to acid than exponential phase bacteria. M. avium incubated with acid in the presence of water was significantly more resistant to pH 2.2 than M. avium in the presence of buffer. Pre-adaptation in water prior to exposure to acidic conditions was also associated with increased resistance to pH 2.2. Isoosmolarity of Hank's balanced salt solution appears to be responsible for the impaired resistance to acid between 2 and 24 h of incubation. These findings indicate that M. avium is naturally tolerant to pH<3 and that pre-adaptation under conditions similar to the conditions where M. avium is found in the environment results in increased acid resistanc

Secreted Mycobacterium tuberculosis Rv3654c and Rv3655c Proteins Participate in the Suppression of Macrophage Apoptosis

Inhibition of macrophage apoptosis by Mycobacterium tuberculosis has been proposed as one of the virulence mechanisms whereby the pathogen avoids the host defense. The mechanisms by which M. tuberculosis H37Rv strain suppress apoptosis and escapes human macrophage killing was investigated.The screening of a transposon mutant bank identified several mutants, which, in contrast to the wild-type bacterium, had impaired ability to inhibit apoptosis of macrophages. Among the identified genes, Rv3659c (31G12 mutant) belongs to an operon reminiscent of type IV pili. The Rv3654c and Rv3655c putative proteins in a seven-gene operon are secreted into the macrophage cytoplasm and suppress apoptosis by blocking the extrinsic pathway. The operon is highly expressed when the bacterium is within macrophages, compared to the expression level in the extracellular environment. Rv3654c recognizes the polypyrimidine tract binding Protein-associated Splicing Factor (PSF) and cleaves it, diminishing the availability of caspase-8. While M. tuberculosis inhibits apoptosis by the extrinsic pathway, the pathogen does not appear to affect the intrinsic pathway. Inactivation of the intrinsic pathway by pharmacologic agents afftects M. tuberculosis and induces cell necrosis. Likewise, inactivation of PSF by siRNA significantly decreased the level of caspase-8 in macrophages.While M. tuberculosis inhibits the extrinsic pathway of apoptosis, it appears to activate the intrinsic pathway leading to macrophage necrosis as a potential exit strategy

MAP1203 Promotes Mycobacterium avium Subspecies paratuberculosis Binding and Invasion to Bovine Epithelial Cells

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease, chronic and ultimately fatal enteritis that affects ruminant populations worldwide. One mode of MAP transmission is oral when young animals ingest bacteria from the collostrum and milk of infected dams. The exposure to raw milk has a dramatic impact on MAP, resulting in a more invasive and virulent phenotype. The MAP1203 gene is upregulated over 28-fold after exposure of the bacterium to milk. In this study, the role of MAP1203 in binding and invasion of the bovine epithelial cells was investigated. By over-expressing the native MAP1203 gene and two clones of deletion mutant in the signal sequence and of missense mutations changing the integrin domain from RGD into RDE, we demonstrate that MAP1203 plays a role in increasing binding in more than 50% and invasion in 35% of bovine MDBK epithelial cells during early phase of infection. Furthermore, results obtained suggest that MAP1203 is a surface-exposed protein in MAP and the signal sequence is required for processing and expression of functional protein on the surface of the bacterium. Using the protein pull-down assay and far-Western blot, we also demonstrate that MAP1203 interacts with the host dihydropyrimidinase-related protein 2 and glyceraldehyde 3-phosphate dehydrogenase proteins, located on the membrane of epithelial cell and involved in the remodeling of the cytoskeleton. Our data suggests that MAP1203 plays a significant role in the initiation of MAP infection of the bovine epithelium

No Holes Barred: Invasion of the Intestinal Mucosa by Mycobacterium avium subsp. paratuberculosis

The infection biology of Mycobacterium avium subsp. paratuberculosis has recently crystallized, with added details surrounding intestinal invasion. The involvement of pathogen-derived effector proteins such as the major membrane protein, oxidoreductase, and fibronectin attachment proteins have been uncovered. Mutations constructed in this pathogen have also shed light on genes needed for invasion. The host cell types that are susceptible to invasion have been defined, along with their transcriptional response. Recent details have given a new appreciation for the dynamic interplay between the host and bacterium that occurs at the outset of infection. An initial look at the global expression pathways of the host has shown a circumvention of the cell communication pathway by M. avium subsp. paratuberculosis, which loosens the integrity of the tight junctions. We now know that M. avium subsp. paratuberculosis activates the epithelial layer and also actively recruits macrophages to the site of infection. These notable findings are summarized along with added mechanistic details of the early infection model. We conclude by proposing critical next steps to further elucidate the process of M. avium subsp. paratuberculosis invasion.Keywords: Fibronectin attachment protein, Infection, Activates dendritic cells, Johnes disease, Enterocytes, SSP paratuberculosis, Peyers patches, Bovine epithelial cells, Immune responses, Cattl

Mycobacterium avium Biofilm Attenuates Mononuclear Phagocyte Function by Triggering Hyperstimulation and Apoptosis during Early Infection

Mycobacterium avium subsp. hominissuis is an opportunistic human pathogen that has been shown to form biofilm in vitro and in vivo. Biofilm formation in vivo appears to be associated with infections in the respiratory tract of the host. The reasoning behind how M. avium subsp. hominissuis biofilm is allowed to establish and persist without being cleared by the innate immune system is currently unknown. To identify the mechanism responsible for this, we developed an in vitro model using THP-1 human mononuclear phagocytes cocultured with established M. avium subsp. hominissuis biofilm and surveyed various aspects of the interaction, including phagocyte stimulation and response, bacterial killing, and apoptosis. M. avium subsp. hominissuis biofilm triggered robust tumor necrosis factor alpha (TNF-α) release from THP-1 cells as well as superoxide and nitric oxide production. Surprisingly, the hyperstimulated phagocytes did not effectively eliminate the cells of the biofilm, even when pre-stimulated with gamma interferon (IFN-γ) or TNF-α or cocultured with natural killer cells (which have been shown to induce anti-M. avium subsp. hominissuis activity when added to THP-1 cells infected with planktonic M. avium subsp. hominissuis). Time-lapse microscopy and the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay determined that contact with the M. avium subsp. hominissuis biofilm led to early, widespread onset of apoptosis, which is not seen until much later in planktonic M. avium subsp. hominissuis infection. Blocking TNF-α or TNF-R1 during interaction with the biofilm significantly reduced THP-1 apoptosis but did not lead to elimination of M. avium subsp. hominissuis. Our data collectively indicate that M. avium subsp. hominissuis biofilm induces TNF-α-driven hyperstimulation and apoptosis of surveilling phagocytes, which prevents clearance of the biofilm by cells of the innate immune system and allows the biofilm-associated infection to persist

Antibodies against invasive phenotype-specific antigens increase Mycobacterium avium subspecies paratuberculosis translocation across a polarized epithelial cell model and enhance killing by bovine macrophages

Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a severe chronic enteritis which affects large populations of ruminants globally. Prevention strategies to combat the spread of Johne's disease among cattle herds involve adhering to strict calving practices to ensure young susceptible animals do not come in contact with MAP-contaminated colostrum, milk, or fecal material. Unfortunately, the current vaccination options available are associated with high cost and suboptimal efficacy. To more successfully combat the spread of Johne's disease to young calves, an efficient method of protection is needed. In this study, we examined passive immunization as a mode of introducing protective antibodies against MAP to prevent the passage of the bacterium to young animals via colostrum and milk. Utilizing the infectious MAP phenotype developed after bacterial exposure to milk, we demonstrate that in vitro opsonization with serum from Johne's-positive cattle results in enhanced translocation across a bovine MDBK polarized epithelial cell monolayer. Furthermore, immune serum opsonization of MAP results in a rapid host cell-mediated killing by bovine macrophages in an oxidative-, nitrosative-, and extracellular DNA trap-independent manner. This study illustrates that antibody opsonization of MAP expressing an infectious phenotype leads to the killing of the bacterium during the initial stage of macrophage infection.This document is protected by copyright and was first published by Frontiers. All rights reserved. It is reproduced with permission. This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by Frontiers. The published article can be found at: http://journal.frontiersin.org/journal/cellular-and-infection-microbiologyKeywords: intestinal tract, M. avium paratuberculosis, antibodies, invasion, macrophage killin

Identification of Mycobacterium avium genes associated with resistance to host antimicrobial peptides

Antimicrobial peptides are an important component of the innate immune defense. Mycobacterium avium subsp hominissuis (M. avium) is an organism that establishes contact with the respiratory and gastrointestinal mucosa as a necessary step for infection. M. avium is resistant to high concentrations of polymyxin B, a surrogate for antimicrobial peptides. To determine gene-encoding proteins that are associated with this resistance, we screened a transposon library of M. avium strain 104 for susceptibility to polymyxin B. Ten susceptible mutants were identified and the inactivated genes sequenced. The greatest majority of the genes were related to cell wall synthesis and permeability. The mutants were then examined for their ability to enter macrophages and to survive macrophage killing. Three clones among the mutants had impaired uptake by macrophages compared to the wild-type strain, and all ten clones were attenuated in macrophages. The mutants were shown also to be susceptible to cathelicidin (LL-37), in contrast to the wild-type bacterium. All but one of the mutants were significantly attenuated in mice. In conclusion, this study indicated that the M. avium envelope is the primary defense against host antimicrobial peptides

Flux Balance Analysis with Objective Function Defined by Proteomics Data-Metabolism of Mycobacterium tuberculosis Exposed to Mefloquine

We present a study of the metabolism of the Mycobacterium tuberculosis after exposure to antibiotics using proteomics data and flux balance analysis (FBA). The use of FBA to study prokaryotic organisms is well-established and allows insights into the metabolic pathways chosen by the organisms under different environmental conditions. To apply FBA a specific objective function must be selected that represents the metabolic goal of the organism. FBA estimates the metabolism of the cell by linear programming constrained by the stoichiometry of the reactions in an in silico metabolic model of the organism. It is assumed that the metabolism of the organism works towards the specified objective function. A common objective is the maximization of biomass. However, this goal is not suitable for situations when the bacterium is exposed to antibiotics, as the goal of organisms in these cases is survival and not necessarily optimal growth. In this paper we propose a new approach for defining the FBA objective function in studies when the bacterium is under stress. The function is defined based on protein expression data. The proposed methodology is applied to the case when the bacterium is exposed to the drug mefloquine, but can be easily extended to other organisms, conditions or drugs. We compare our method with an alternative method that uses experimental data for adjusting flux constraints. We perform comparisons in terms of essential enzymes and agreement using enzyme abundances. Results indicate that using proteomics data to define FBA objective functions yields less essential reactions with zero flux and lower error rates in prediction accuracy. With flux variability analysis we observe that overall variability due to alternate optima is reduced with the incorporation of proteomics data. We believe that incorporating proteomics data in the objective function used in FBA may help obtain metabolic flux representations that better support experimentally observed features