Study of MAP Proteins

The genome of MAP has been sequenced and available for 15 years (Li et al., 2005). A dozen more genomes of MAP have since followed including bovine isolates (Amin et al., 2015; Mobius et al., 2017), ovine isolates in the USA and Australia (Bannantine et al., 2012; Brauning et al., 2019), camel isolates (Ghosh et al., 2012) and human isolates (Wynne et al., 2011; Bannantine et al., 2014b).

These genome sequences are discussed in depth in Chapter 6 of this book. With these genome sequences in hand, the initial strategy to identify diagnostic antigens was to perform a comparative genomics analysis and select those genes that are present only in MAP. Then those MAP-specific genes were expressed and analysed for antigenicity. However, this strategy yielded a limited number of genes due to the very high sequence identity between MAP and other members of the M. avium subspecies. Most of the unique genes resided in large-sequence polymorphisms (Semret et al., 2005). Unfortunately, while some of these MAPspecific gene products did show immunoreactivity (Paustian etal., 2004; Dernivoix etal., 2017), none of these ultimately were demonstrated as a discriminating antigen that could consistently distinguish infected from non-infected animals. This finding meant that the specificity part of the equation could not be satisfied simply because the sensitivity part of the equation was low for that group of proteins. Researchers have acknowledged this fact and either looked for strong antigens that are not necessarily unique to MAP, or looked deeper for MAP-specific epitopes within otherwise conserved mycobacterial proteins. An example of this is found in the gene designated MAP_1025 (encoding an RDD family protein), which does contain an epitope that only exists in MAP strains (Bannantine et al., 2011). The specificity of this seven-amino acid epitope is due to a single non-synonymous nucleotide polymorphism that occurs only in MAP.

Other such examples likely exist, but their diagnostic utilities have yet to be discovered.

Current strategies for identifying diagnostic antigens are more global on the whole pro- teome scale as opposed to focusing on just a few MAP-specific proteins. Hughes et al. examined proteome expression differences between MAP and M. avium subspecies avium to find 32 MAP proteins that did not appear to be expressed by M. avium avium in the same in vitro culture conditions (Hughes et al., 2008). However, when two of these proteins were incorporated into an IFN-γ release assay, they could not distinguish between MAP and M. avium avium infected cows (Hughes et al., 2017). Still another approach is to examine lipid antigens of MAP. There are known differences in the lipid profiles of MAP and other M. avium subspecies, and the characterized MAP-specific lipopeptides are antigenic (Biet et al., 2008; Mitachi et al., 2016).

The study of MAP proteins should not be solely diagnostic focused. There is a lot of interesting biology associated with this pathogen that could yield clues to its pathogenicity. However, even with additional genome sequences and their more recent annotations, MAP still has an abundance of proteins with unknown function, which are termed hypothetical proteins. Approximately 70.4% of the MAP genome is annotated as hypothetical (unknown function, no similarity) or conserved hypothetical proteins (unknown function, but similar to other proteins in a database). This is considerably more than the 25.9% hypothetical proteins assigned for the first bacterial genome sequenced, Haemophilus influenzae, which sets the standard among the best-characterized bacteria (Shahbaaz et al., 2013). More biochemical and proteomic studies are needed to functionally characterize the MAP proteins categorized as hypothetical.

8.2.1 Functional characterization of MAP proteins

Other than the obvious BLAST (Basic Local Alignment Search Tool) search approach (Altschul et al., 199 7) which is already used to annotate genomes, one way to begin functionally characterizing proteins is to identify how they interact with their environment.

Proteins do not act in isolation, but interact with each other to perform specific functions. Cell development and division, transcription and translation, transport and metabolism can be defined by the activity of protein complexes. Knowing which proteins are assembled in these complexes provides critical clues to the function of these proteins. Non-denaturing (native) gel electrophoresis enables the separation and analysis of protein complexes on a proteome-wide scale. Seven MAP membrane protein complexes have been isolated using this method and their subunits defined (Leite et al., 2015). Complexes II and VII each had nine subunit proteins identified by mass spectrometry. In complex I, the major membrane protein (MAP_2121 c) was present along with a cysteine desulfurase (MAP_2120c), an enzyme that removes sulfur from cysteine for the biosynthesis of co-factors. Although this major membrane protein has long been known as a virulence protein (Bannantine et al., 2003) and antigen (Triccas et al., 1996), its association with cysteine desulfurase may suggest a biosynthetic function as well. Other proteins present within complex II (linocin and a peroxidase) were found to have strong interactions predicted by String analysis (Szklarczyk etal., 2015). These interactions should be confirmed by Far western blot analysis (Wu et al., 2007) or the older two hybrid strategies (Fields and Sternglanz, 1994). One MAP protein, encoded by MAP_1203, has been shown to interact with bovine epithelial cell proteins using the Far western blot method (Everman et al., 2018). Initially annotated as a hypothetical protein, we now know it is surface located, promotes invasion and interacts in a specific way with the host.

Protein location on the bacterial cell is another potential clue to understanding function. For example, proteins in the cell wall are unlikely to affect DNA repair or regulate transcription, but rather might be involved in export or transport functions. The predicted locations of MAP proteins are shown in Table 8.1 and it is noteworthy that 1562 proteins could not be located based on this bioinformatic topology analysis (Yu et al., 2010).

Extracellular or secreted proteins have been considered antigenic (Lanigan et al., 2007; Shin et al., 2010) and MAP has 82 such proteins. The majority of proteins (1857) were located in the cytoplasm (Table 8.1). However, a surface exposed protein that is a strong antigen is likely the ideal diagnostic antigen. An understanding of protein, glycoprotein or lipoprotein topology within the cell wall and whether it protrudes from the outer cell wall layer to become surface exposed is generally lacking. A major effort directed at this goal of defining surface exposure of MAP proteins was by He and De Buck

Table 8.1. Predicted location of Mycobacterium avium subsp. paratuberculosis (MAP) proteins based on PSORTb 3.0 analysis.

Location	No. of proteins^a	% of proteome	Ave score^b
Cytoplasm	1857	42.2	9.28
Extracellular	82	1.9	9.62
Membrane	833	18.9	9.66
Periplasm	60	1.4	9.69
Outer	7	0.1	9.67
membrane
Unknown	1562	35.5	2.29

^aTotal number of proteins in the MAP proteome is 4401. ^bPSORTb final prediction score ranges between 1 (low probability) to 10 (high probability). Score must be above 7.5 to make a location prediction (Yu et al, 2010).

(2010). They performed a trypsin shaving method on the cell wall of intact, viable bacteria and identified 38 proteins that are surface exposed. These proteins are at the front line for adhesion, invasion, cell division or perhaps other interactions with the host cells during infection.

8.2.2 MAP lipoproteins

Of the MAP proteins that are known, 63 are annotated as lipoproteins, which are considered fertile ground for strong antigen discovery based on studies with Mycobacterium tuberculosis (Becker and Sander, 2016). The outer layers of the mycobacterial cell envelope are composed of at least three types of non-covalently associated glycolipids interlaced in a carbohydrate matrix. The glycolipid types include phenolic glycolipids, glycopeptidolipids (GPLs) and lipooligosac- charides. Interestingly, MAP does not produce GPLs like the other M. avium subspecies. Instead, it produces unique lipopeptides, which include Para-LP-01, a lipopentapeptide in the bovine strains (Eckstein et al., 2006) and a lipotripeptide in the ovine strains (Bannantine et al., 2017b). The Para-LP-01, also known as L5P, elicits an antibody response in cattle and sheep (Biet et al., 2008; Thirunavukkarasu et al., 2013) as well as humans (Verdier et al., 2013), but not a cell-mediated immune response (Souriau et al., 2017). In addition, the abundance of this lipid molecule decreases during infection (Everman et al., 2015) and this, along with other documented lipids (Alonso-Hearn et al., 2010) and fatty acid changes (Alonso-Hearn et al., 2017), may contribute to the severe inflammation of the bovine intestine.

GPLs and lipopeptides are synthesized by non-ribosomal peptide synthetases that are encoded by large genes with a modular organization. Transposon mutagenesis of one non-ribosomal peptide synthetase, pstA (MAP_1242), changed the 2-D lipid profile of MAP as analysed by thin-layer chromatography (Wu et al., 2009). Characterization of the missing lipid in the pstA mutant suggested it was a lipotripeptide since this gene encodes three amino acid modules, which differentiates it from M.

avium (encoding two modules) and Mycobacterium smegmatis (encoding four modules). Phenotypically, the pstA knockout mutant could not produce an extracellular matrix important for biofilm formation and was not able to transverse the intestine as efficiently as the wild type. As mentioned above, another lipotripeptide, termed L3P, was discovered uniquely in ovine strains of MAP. The non-ribosomal peptide portion of this molecule is encoded by MAP_1420 and its structure was characterized in detail to reveal the tripeptide Phe-N-Methyl-Val-Ala attached to a lipid moiety (Bannantine et al., 2017b). Still another MAP lipopeptide, isolated from an ethanol extract of MAP (Eda et al., 2006), has been biochemically characterized, shown to be immunogenic and defined to five amino acids (Mitachi et al., 2016). Given that the genes required for the biosynthesis of these unique peptide-lipid cell wall structures are 20 kb in length, and thus take enormous amounts of energy to replicate, it is hypothesized that they must provide critical roles. The MAP lipoarabi- nomannan molecule has largely been ignored this past decade except for a vaccination study (Jolly et al., 2016) and an examination of its immunostimulatory properties on bovine macrophages (Souza et al., 2013).

8.2.3 MAP virulence proteins

A number of virulence proteins have been proposed despite the lack of proteome-wide screens for this category of proteins. Transcriptional regulators, such as the sigma factors SigH (MAP3324c) and SigL (MAP4201) result in attenuation when they are deleted, leading investigators to infer that they may control the expression of virulence genes (Ghosh et al., 2013, Ghosh et al., 2014). SigH in particular is important for MAP survival in IFN-γ activated bovine macrophages (Ghosh et al., 2013). Likewise, the LuxR transcriptional regulator (MAP1875c) is expressed at higher levels when MAP is exposed to milk (Alonso-Hearn et al., 2010). This protein has an ATPase domain and it regulates four lipid biosynthesis genes and two invasion genes along with five other upregulated genes. Despite these notable studies, very few MAP virulence genes have been examined in depth and even less have been confirmed by knockout mutation experiments, which are not trivial in MAP (Park et al., 2008; Scandurra et al., 2010). None the less, a handful of knockout mutations have shown attenuation in either macrophages or animal models (Scandurra et al., 2009; Bannantine et al., 2014a; Ghosh et al., 2014; Meissner et al., 2014).

Several MAP virulence proteins are involved in binding and/or invasion of bovine epithelial cells, which represent the initial infection event in paratuberculosis. In addition to the 10 MAP proteins discussed previously (Bannantine and Bermudez, 2013), MAP_1203 also plays a role in epithelial cell invasion (Everman et al., 2018). Transcription of this gene is increased 28-fold when MAP is exposed to milk. The gene product contains a signal sequence that guides the protein into the MAP cell wall where it interacts specifically with two host proteins, dihydropyrimidinase-related protein 2 and glyceraldehyde 3-phosphate dehydrogenase, at the epithelial cell membrane. Other virulence proteins include a membrane serine protease (MAP_0403), which is upregulated in cultured macrophages and epithelial cells as well as under in vitro acid conditions (Kugadas et al., 2016). Thus MAP_0403 imparts acid stress tolerance to MAP, but was not detected in oxidative or nitrogen stress conditions (Kawaji et al., 2010). And finally, the mptD gene (MAP_3733 c) present on the MAP-specific large-sequence polymorphism 14 is important for survival in the early macrophage environment and is necessary for virulence in the mouse model (Meissner et al., 2014).

8.3

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Source: Behr Marcel A., Stevenson K., Kapur V. (eds.). Paratuberculosis: Organism, Disease, Control. 2nd edition. — CAB International,2020. — 439 p.. 2020

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