Pathogenic mechanisms and etiologic aspects of Mycobacterium avium subspecies paratuberculosis as an infectious cause of cutaneous melanoma

Infectious etiologies have previously been proposed as causes of both melanoma and non-melanoma skin cancer. This exploratory overview explains and presents the evidence for the hypothesis that a microorganism excreted in infected ruminant animal feces, Mycobacterium avium subspecies paratuberculosis (MAP), is the cause of some cases of cutaneous melanoma (CM). Occupational, residential, and recreational contact with MAP-contaminated feces, soil, sand, and natural bodies of water may confer a higher rate of CM. Included in our hypothesis are possible reasons for the differing rates and locations of CM in persons with white versus nonwhite skin, why CM develops underneath nails and in vulvar skin, why canine melanoma is an excellent model for human melanoma, and why the Bacille Calmette-Guérin (BCG) vaccine has demonstrated efficacy in the prevention and treatment of CM. The pathogenic mechanisms and etiologic aspects of MAP, as a transmittable agent underlying CM risk, are carefully deliberated in this paper. Imbalances in gut and skin bacteria, genetic risk factors, and vaccine prevention/therapy are also discussed, while acknowledging that the evidence for a causal association between MAP exposure and CM remains circumstantial.


| INTRODUCTION
[7] Known nonsolar risk factors for CM include chemicals such as pesticides 8 and genetic conditions. 9CM often occurs in sites where the sun does not shine directly 10 and moderate sun exposure is protective rather than detrimental. 11Indoor occupations generally confer a higher versus lower risk for CM. 12,13Another suspected nonsolar cause of human CM is the animal microorganism MAP.Besides MAP, exposure to other infectious agents, including human endogenous retroviruses 14,15 and human papillomaviruses, 10,16,17 may contribute to the development of CM.Herein, we discuss the pathogenic characteristics and etiologic features of MAP exposure in the context of CM.Discourse is also focused on imbalances in gut and skin bacteria, genetic risk factors, and vaccine prevention/therapy, laying the groundwork for future hypotheses and research.

| Bacteria-related inflammation
The bacterial parasite MAP causes a chronic inflammatory enteropathy called Johne's disease or paratuberculosis 18 in large domestic ruminants such as dairy cattle and beef cattle, 19 smaller domestic ruminants such as goats and sheep, 20 wild ruminants and other nondomestic animals such as badgers, coyotes, crows, opossums, cats, rabbits, racoons, 21 and primates. 22MAP also infects nonruminant animals including humans and is considered to be an "endemic trans-species pathogen". 231][32][33][34] One may posit that chronic inflammation is a predecessor event for many cancers including melanoma.

| A role for MAP
A role for MAP in the development of CM previously has not been proposed, but is suggested by the following epidemiologic characteristics of CM.MAP is heavily excreted in an infected animal's feces, and animal microorganisms excreted in an infected animal's feces have been discussed as possible causes of farming and agriculture-associated cancers. 35,36With rare exceptions, 37 a majority of studies describe an increased rate of CM in farmers and agricultural workers, [38][39][40][41][42][43][44][45][46][47] although uncertainty exists regarding the strength of the associations.Studies have discussed that there is an excess of CM specifically in the faces, necks, and scalps of farmers 48,49 ; three locations where MAP contaminated feces could easily reach otherwise clothed farmers.Solar radiation also may be an explanatory factor for these locations. 50few studies show an increased rate of CM especially in cattle farmers.One study showed female dairy workers had the fifth highest occupational rate of CM. 51 A majority of CM patients in two African studies were farmers or cattle herders. 52,53However, this could merely reflect the main occupation of the underlying population.
Veterinarians have an increased risk of CM [54][55][56][57] and animal microorganisms have been proposed as possible causes for cancers identified in this occupational group. 35,46,55,58Two studies differentiated between "large animal" and "small animal" veterinarians, showing an increased risk of CM in the former. 55,59One study showed a high standardized mortality ratio for CM in large animal veterinarians, with cases occurring before the age of 30. 60airy and beef cattle are the primary animals that may be responsible for putting these types of veterinarians at an increased risk for CM.
Increased rates of CM in specific countries and regions within countries can be correlated with the presence of four main domestic ruminants: dairy and beef cattle, goats, and sheep.These animals often are located near bodies of water where their manure runoff causes water contamination.Higher rates of CM also have been observed in specific areas of the United States associated with considerable numbers of dairy cattle.Utah, Minnesota, Vermont, Idaho, and Iowa have the highest incidence rates of CM in the United States. 613][64][65][66] The number of dairy animals per farm ranges from 200 to over 15,000 animals.All dairy operations in the United States with over 200 animals have at least one animal heavily excreting MAP organisms in its feces. 67This translates to significant amounts of exposure to MAP bacteria among residents in the vicinity of these farms (Figure 2). 68,69In Idaho, there has been a threefold increase in the incidence of CM 70 associated with the development of CAFOs. 71[75] Furthermore, spatial clustering of CM near concentrations of dairy cattle with high rates of MAP infections has been documented in British Columbia, Canada. 76,77There are spatial clusters of high rates of CM in Ecuador 78 associated with proximity to dairy cattle with high prevalence rates of MAP infection. 79The higher rate of CM in northern versus southern Italy corresponds with the increased concentration of dairy cattle in several northern provinces. 80A high rate of CM in southern Brazil 81 is associated with prevalent dairy cattle farming. 82 theory, the high rate of CM among Australians may be explained not only by exposure to UV radiation but also exposure to sand and water contaminated by the feces of beef and dairy cattle and sheep.An increased rate of CM in coastal areas of Australia compared with inland areas, 83 specifically in Queensland, 84 overlaps with the 28 million beef cattle, 85 1.6 million dairy cattle, 86 and 104 million sheep 87 in Australia.These are concentrated along the coastlines in Queensland and New South Wales near swimming and surfing areas. 88,89ne study localized the highest rate of CM to a specific section of southeast Queensland, precisely where the highest concentration of beef and dairy cattle are located. 90P organisms have been documented to remain viable in soil and dirt for over 1 year. 91,92n countries where MAP infections of domestic livestock are longstanding, MAP is present in soil samples throughout the country. 93The presence of MAP in soil and dirt may explain the increased risk of CM in chimney sweeps, specifically in their upper limbs, suggesting "skin contact with a harmful agent" 94 and may underlie the increased risk of CM in outdoor athletes and sportsmen. 95As well, soil, 96,97 sand, 98,99 and natural bodies of water 100 contaminated with MAP through manure runoff may explain the higher rate of CM in people exposed intermittently or occasionally to sunlight. 84,101elanoma is not considered to be associated with chronic sun exposure, but rather with heavy blistering overdoses, and is more common among non-manual workers of higher social status."47 It may not be sunlight they are being occasionally or intermittently exposed to, but rather coming into contact with MAP through the activities they engage in when they are occasionally outside, including lying on and playing in MAP contaminated sand and water.This may explain the increased incidence of CM associated with "holidays spent at the beach."95 Nonetheless, comparative studies are needed of CM in areas without heavy concentrations of cows.
Painful or blistering sunburns [102][103][104] may increase the risk of CM because sunburn damages the epidermal layer of the skin, allowing MAP contaminated soil, sand, or water direct access to the melanocytes located in the epidermal-dermal junction.The association of trauma with melanomas may also be related to a similar break in the epidermal layer of the skin. 105,106ing to MAP's hydrophobic cell wall, all the MAP organisms in a body of water are concentrated at the surface. 107,1080][111] The concentration of MAP, first at the surface of natural bodies of water and then their further concentration by aerosolization, is consistent with the increased rate of CM associated with occupational or recreational activities at the surface of bodies of water, including swimming, 112,113 surfing, 113,114 boating, 101,115,116 and fishing. 101A recent study showing a 96-fold increased rate of CM in surfers (vs.an 18-fold increased rate in swimmers) 113 is consistent with the forceful injection of aerosolized, concentrated MAP organisms into the skin.Alternatively, surfers might be more exposed to the sunlight/ reflective rays while standing on their surfboards (vs.swimmers being more underwater).Questionably, CM rates may not be lower among surfers in areas with fewer cattle.
Several hypotheses have been proposed for the increased rate of CM in swimmers, including chlorine disinfection byproducts 116,117 and UV radiation's effect on organic matter creating hydrogen peroxide, which then damages hair follicles. 118An alternative explanation is the presence of MAP in both natural bodies of water contaminated with manure runoff, 100 as well as in chlorinated potable water. 119MAP is known to be completely impervious to chlorine disinfection 120 and is present in almost 90% of tap water samples in the United States. 121e presence of MAP in chlorinated water may explain the existence of vulvar melanomas, which are a subset of CM. 122 It is difficult to see how UV radiation penetrates to the vulvar tissues, but very easy to see how MAP-contaminated chlorinated water does.One example is a case where a 68-year old woman with a vulvar melanoma 123 reports over 900 h of swimming in indoor chlorinated water swimming pools, the bulk of which occurred before the age of 16 (Laura S. Brown, Ph.D., email communication, January 16, 2023).A common location of vulvar melanomas in the clitoris 124 may be explained by the ability of MAP-contaminated chlorinated water to access the space underneath the hood of the clitoris.UVB (medium wavelength, 280-315 nm) radiation is completely unable to penetrate the nail plate and UVA (longest wavelength, 315-400 nm) radiation only minimally. 125However, the ability of MAP-contaminated soil, dirt, and MAP-contaminated water, either chlorinated or natural water, to get underneath the nails, may explain the occurrence of subungual melanomas.
Metalworking fluids that are contaminated with MAP 126,127 may explain the increased incidence of CM in the necks of welders, 128 as the neck is where the spray of metalworking fluid reaches the skin.The increased risk of CM in petroleum workers and printers 129 may be explained by the large amounts of possibly MAP-contaminated water used for crude oil extraction 130,131 and in printing processes. 132Again, details pertaining to the exact levels of contamination in these studies are equivocal.
Hair cover may protect against invasive melanoma of the head, neck, and ear 133 because MAP organisms may be clinging to the hair, rather than reaching the scalp, neck, or ear skin.Some studies have shown that obesity increases the risk of CM, specifically through increased body surface area rather than body mass index, perhaps because having more skin surface area increases the chances of MAP penetrating the skin. 134,135The increase in skin surface area with increasing height may also explain the putative association of CM risk with increasing childhood height. 136,137As well, the increase in surface area could be associated with both increased sun exposure and increased exposure to MAP.Potential alternative pathways of MAP exposure are summarized in Figure 3.

| Skin color
Individuals with white skin 138 have a higher rate of CM, while individuals with nonwhite skin have a lower rate of CM.The location of CM in individuals with nonwhite skin differs from the location of CM in individuals with white skin.In insects, the production of melanin (melanization) is a response to bacterial penetration of the cuticle of the exoskeleton, and encapsulates the invading bacteria. 139Authors have suggested that human skin also acts as an antimicrobial barrier, 140,141 and skin color has developed as a protective factor against microorganisms. 141,142"Animal husbandry practices" have been suggested as one pressure on the development of skin color. 141Skin color was a protective factor among Vietnam Veterans regarding bacterial and parasitic infections.Black Vietnam Veterans were less likely to develop such infections in comparison to white Vietnam Veterans. 141e study has shown the individual melanosomes that are the predominant type of melanosome in nonwhite skin are bacterial lysosomal degradative organelles, in contrast to the melanosome clusters in white skin that have no effect on bacteria. 143Darker skin tone corresponds to larger melanosomes and more effective antibacterial degradative organelle melanosomes. 144Individual melanosomes in hair follicles increase in size as a person ages. 145The possibility that individual melanosomes in skin may be smaller in children in contrast to adults may explain the greater risk of CM in children with nonwhite skin than adults with nonwhite skin. 146An alternative explanation is that children of any skin color are more likely to play in dirt than adults.
Individuals with nonwhite skin rarely develop CM.Only 1237 Asians with CM were identified in a United States database versus 409,000 non-Hispanic whites over the same time period. 147Another study identified over 48,000 non-Hispanic whites with CM versus 932 Hispanics whites, 394 Asian Americans and Pacific Islanders, 251 African Americans, and 52 Indigenous (First Nations) people. 138The only nonwhite skin that contains predominantly melanosome clusters are the palms of the hands and soles of the feet. 148This may explain the almost exclusive locations of these two CM sites in individuals with nonwhite skin. 149Both white and nonwhite individuals develop palmar and plantar melanomas, 150 but nonwhite individuals rarely develop CM in any other location.
The increased rate of vulvar melanomas in white versus black women 151 possibly suggests that the labial skin of black women may be another skin site that contains predominantly melanosome clusters rather than individual melanosomes.

| Additional considerations of MAP exposure
Farmers and swimmers are exposed to MAP in outdoor environments and are exposed to sunlight as well as MAP.UV radiation from sunlight may be immunosuppressive and indirectly increase the risk of infection with MAP. 152Conversely, UV radiation may directly cause melanoma independent of an MAP infection by means of systemic alteration in the immune system. 153,154Some alternative sources of UV radiation in addition to sunlight include tanning beds, artificial lighting (mercury vapor, halogen, fluorescent, and incandescent), and lasers. 155e exposure of farmers to pesticides has been implicated in their increased rate of cancer. 1569][160] However, we are not aware on any studies showing decreased CM risk with herbicides killing pathogenic bacteria.
In some cases, a direct, positive association may not exist for MAP exposure.For example, there is a reduced rather than increased incidence of CM in butchers presumably exposed to MAP contaminated meat and blood. 161Also, the association of CM with the leather industry is weak and has been linked to the chemicals used in leatherwork rather than exposure to animal microorganisms. 162,163

| How MAP could cause CM
5][166] Furthermore, it has been shown that Mycobacterium tuberculosis (MTB), with features similar to MAP, may promote oncogenesis through chronic inflammation resulting from increased levels of tumor necrosis factor-alpha (TNFα). 167MAP, like MTB, is a persistent and asymptomatic infection. 168Approximately eight of nine individuals remain MAP positive 1 year after their first positive test. 168 propose an additional specific mechanism for MAP-associated CM.Recently, it has been observed that melanocytes are the precursor cells for CM and CM cells are malignant melanocytes. 169Conceivably, malignant melanoma cells are melanocytes that are chronically infected with MAP.Chronic MAP infection of cutaneous melanocytes may result in CM by the continuing secretion of osteopontin by MAP-infected melanocytes.Osteopontin is a glycoprotein secreted by cells infected with mycobacteria of different species.These species include MAP, [170][171][172] Mycobacterium avium intracellulare, 173 MTB, 174 and the Bacille Calmette-Guérin (BCG) vaccine 173,175 (an inactivated strain of Mycobacterium bovis).
Serum osteopontin levels are increased in patients with CM. 176 Osteopontin enhances the host cell's response to the infecting Mycobacterium by stimulating T helper cell type-1 secretion. 173Osteopontin is thought to stimulate epithelial to mesenchymal transition. 177,178steopontin also has antiapoptotic effects in melanocytes. 179It is possible that a melanocyte infected with MAP is chronically secreting osteopontin and that persistent osteopontin antiapoptotic stimulation results in the malignant transformation of infected melanocytes. 179egional and distant metastases of CM may be equivalent to lymphatic and hematogenous dissemination respectively of MAP-infected melanocytes. 180P organisms typically reach melanocytes from the skin surface, rather than from an internal location.The increased rate of CM in patients with idiopathic inflammatory bowel disease, particularly Crohn's disease, 181,182 may be explained by skin contact with rather than ingestion of MAP-contaminated soil, unchlorinated water, or chlorinated drinking water.The ingestion of chlorinated tap water has been linked to a cluster of Crohn's disease, 183 but individuals with inflammatory bowel disease also are bathing and swimming in MAP-contaminated chlorinated water.On the other hand, the persistent ingestion and lingering of MAP may disrupt the gut microbiome leading to systemic inflammation and cancer.Additionally, Crohn's disease conceivably could cause "holes" in the internal mucosal lining (similarly to what sunburn does on the surface of skin) that allow MAP from tap water to enter the system in a way that would not be possible with intact mucosa.

| Imbalances in gut and skin bacteria
MAP is believed to readily colonize the gut, as illustrated by its appearance in patients with Crohn's disease, and to a lesser degree in those without this condition. 184This bacterium is notoriously persistent and difficult to clear with anti-MAP antibiotics, often taking many months or years when effective to eradicate the organism from the gut and body.Reported infection and prevalence rates of this nonspore-forming microbe likely are underestimated given the paucity of routine testing and standard testing methodology.Environmental sources for gut MAP are ubiquitous, including the consumption of infected dairy foods, underdone meat, and drinking or encountering infected soil and water.In many cases, the organism is resistant to pasteurization, further exasperating public health control efforts.Various inflammatory skin conditions such as psoriasis, atopic dermatitis, and rosacea have been associated with bacteria in the gut, with skin cancer also hypothesized to belong to this list of disorders. 185Patients with serum bacterial DNA presumed to originate from the intestinal lumen specifically manifest high levels of inflammatory factors (e.g., IL-1β, IL-6, IL-12, tumor necrosis factor, and interferon γ), suggesting a decreased integrity of the gut epithelium and overall negative homeostasis (Figure 4). 186,187e "gut-skin axis," vis-á-vis dysbiosis (microbiota imbalance), has been postulated to underlie CM by impairing the intestinal barrier.This is characterized by systemic inflammation, oxidative stress, and maladaptive redox processes, potentially facilitating tumor progression and acquired chemoresistance of skin cancer. 188Following ingestion, MAP appears to penetrate the intestinal wall by way of the ileal mucosa but also can cross the gut mucosa through enterocytes, independently of Peyer's patches. 189This Grampositive, acid-fast bacterium is resistant to intracellular degradation.Infected macrophages easily spread to the lymphatic system to impair immunologic responses and manifest as increasing amounts of adhesion molecules and the formation of granulomas (e.g., KdpFABC complex). 189MAP can obstruct superoxide anions, hydrogen peroxide, and hydroxyl radicals to affect immune modulation (interleukin-10/interleukin-12, and interleukin-23).The virulence of MAP is intrinsically related to its ability to acquire nutrients within the gut and break down host fatty acids.The long-term survival of MAP within host macrophages is associated with genetic modifications linked to iron limitation and nitric oxide build-up.
Imbalances in gut microbiota have been correlated with incipient melanoma and weakened response to immune therapies. 190Bacterial profiles enable the risk stratification and discrimination of melanoma from non-melanoma, with differences observed in the structure and richness of colonies in patients with early-stage melanoma.Although MAP is not specifically mentioned, similar gut bacteria appears to affect immune surveillance and cancer development.By analogy, one may infer that gut MAP increases the level of circulating pro-inflammatory cytokines and in effect prompt immune cells to migrate toward the skin, resulting in local inflammation, a key step in the neoplastic development of melanocytes.
While the gut represents the primary home of human commensal microbiota, microbial toxins operating within the skin (the largest organ of the body) may facilitate chronic inflammation and cell damage, precipitating melanoma development and progression. 185s the skin comes in direct contact with sunlight, it has been further suggested that the deleterious effect of UV radiation-induced immunosuppression is intensified in the presence of certain cutaneous bacteria.In theory, UV-radiation (a pivotal carcinogen) may interact with MAP to compromise the microenvironment of CM, with important disruptions to CD (cluster of differentiation)8+ T-cells, regulatory T-cells, cytokines, chemokines, and tumorassociated macrophages.If not countered by the presence of "good" bacteria to promote intestinal health and positive vitamin D levels, local UV-induced changes to skin microbiota may exasperate "bad" gut microbiota and vice versa, through systemic pathways. 186veral immunotherapeutic advances, such as anti-programmed cell death 1 (anti-PD-1), anti-programmed death-ligand-1 (anti-PD-L1), and anti-cytotoxic T-lymphocyte-associated antigen 4 (anti-CTLA-4) have become available to better manage moderate to advancedstaged melanoma. 191,192However, the positive response to these compounds is limited to a subsegment of the melanoma population and even then may not be durable over time.One explanation posits that innate and adaptive immunity may be influenced by the gut microbiome, wherein variations in bacteria colonies differentially effect treatment outcomes and frequency of immune-related adverse effects.That is, responders (Rs) versus non-responders (NRs) may manifest differences in terms of their gut microbiome balance.Research efforts aiming to optimally adjust the gut microbiota to improve the therapeutic benefit of these immunologic agents include fecal microbiota transplantation, probiotics, and diet.While such attempts mainly have focused on enriching the microbiota with so-called "good" bacteria, there has been a dearth of research aimed at measures to directly minimize the negative load of harmful bacteria in the gut.One possible avenue for future study would be to assess the potential benefit of RedHill Biopharma (RHB)-104, an anti-MAP mix of antibiotics.While such an approach likely would spill over to eradicate beneficial bacteria colonies, the post-administration of probiotics might be required to replenish healthful bacteria.

| Genetic factors
Approximately 90% of individuals presenting with CM do not have any affected relatives, suggesting that inheritance only plays a minor role in the direct development of melanoma. 9Rather, in most cases, genetics appear to have an indirect role, interacting with environmental factors (possibly MAP) to increase melanoma susceptibility through inflammatory and various other growth pathways.While most genetic variants associated with melanoma risk are somatic in nature, this does not rule out a polygenic mode of inheritance involving the interplay of multiple low-risk alleles or rare mutations in high-penetrance genes (e.g., ACD, BAP1, CDK4, CDKN2A, POT1, TERF2IP, TERT).A link with DNA repair, as suggested for melanocortin 1 receptors (MC1R), may underlie melanoma predisposition, especially in non-pigment pathways (Figure 5).
A recent meta-analysis of 36,760 melanoma cases identified an increased risk for 68 independent SNPs, corresponding to 54 loci. 193Although many of these SNPs were associated with "pigmentation, tanning response, nevus count, and telomere maintenance", a subset of sites diagnosed in populations at low risk for UV-induced melanomas (e.g., nail beds, palms, soles), appear to be unrelated to pigmentation.Furthermore, in an extended pleiotropic analysis, many loci were not associated with nevus count or hair color, suggesting that genetic variants often "act outside of … classic cutaneous melanoma risk phenotypes."Additionally noted, an immunologic role for melanoma risk was supported by the significant genome-wide association with rs28986343 at the HLA locus, in line with previous studies indicating an "association between rs408825 and expression of the innate immunity gene MX2."Interestingly, several risk alleles for vitiligo, an autoimmune melanocyte-related disorder, are protective for CM, highlighting the important role of immunity in the development of CM.
The evasion of immune response has been associated with BRAF mutations "affecting signaling pathways in melanoma development." 194Abnormal BRAF kinase activity occurs in approximately half of CMs owing to point mutations at codon 600, with the replacement of glutamic acid for valine.BRAF mutations often arise in tumors that are free of "chronic sun induced damage."While this raises the possibility of a nonsolar role for MAP in the etiology of melanoma, there are no studies to date that support this supposition.Overall, the immunology of melanoma is complex with many redundant pathways.Targeted immunebased therapy has shown success in a subset of melanoma skin cancer patients.However, acquired resistance is a commonly observed outcome.Independent of UV radiation, CMs often present in mixed cancer syndromes attributable to "mutations in PTEN, BRCA2, BRCA1, RB1, and TP53." 195 The "epithelioid cytology of melanocytic tumors may suggest an underlying BAP1 mutation," frequently characterized as BAP1 tumor predisposition syndrome (BAP1-TPDS).This syndrome is associated with several tumors of nonsolar origin, such as renal cell carcinoma, mesothelioma, and posteriorly occurring uveal melanomas.The nonsolar role of MAP in posteriorly located uveal melanoma has been recently explored in a narrative review on this topic. 34e genetic basis for the survival and persistence of MAP in a host organism is an important requisite for skin cancer development.A recent "Hidden Markov Model" analysis identified 430 in vivo genes essential for colonization in the natural host and 260 in vivo growth-defect genes. 196Furthermore, a gene ontology (GO) enrichment analysis "revealed an overrepresentation of genes for biological process, molecular function, and chemical component categories," with top-ranked GO terms suggesting the involvement of "in vivo essential genes in DNA repair, pathogenesis, symbiosis and parasitism growth, response to stimuli and oxidation-reduction processes."In particular, the transcription initiation factor "RNA polymerase sigma subfactor A (sigA)" was identified an in vitro essential gene, while genes deemed to underlie in vitro growth of MAP and H37Rv included "PurC, PurL, and PurN," vis-á-vis biosynthesis of arginine, aromatic amino acids, ATP, folic acid, purine, and riboflavin.
While the above critique has largely focused on chronic intestinal inflammation attributable to MAP in ruminants, one may hypothesize that a similar set of genes underlie colonization, persistence, and survival of mutant MAP in the gut and skin of human hosts.For example, the upregulation of SigA has been noted as an in vivo growth promoter in the "sister" organism MTB in humans.Similarly, the open reading frame (ORF) Rv031 for human MTB was mentioned as a virulent promoter of "bacilli survival in harsh conditions and inside macrophages."197 Another gene glnB, believed to encode a nitrogen regulatory protein, also has been purported to resist "the IFN-γ-activated macrophage" environment in human MTB, 198 curtailing a key host defense of MTB as a persistent infection.As well, mutations among individuals in the Toll-like receptors (TLRs) manifest increased susceptibility to mycobacteria, wherein TLR9 specifically is believed to "initiate responses that are critical in defense against MAP." 199

| Other means of evading the immune system
As evolutionary genetic processes have allowed MAP to efficiently infect host animals and humans, a cascade of nongenetic mechanisms conceivably are involved as well to evade immune response and dysregulation of cellular metabolism.Of importance, MAP "in human cells are cholesterol dependent" wherein "these bacteria localize to cholesterolrich compartments that are slow to acidify." 200The persistence of MAP allegedly has been linked to interruption of phagosome acidification and the "ineffective recruitment of RAS-associated binding7 (Rab7)-interacting lysosomal protein (RILP) to the phagosomal membrane."Furthermore, MTB Mce3e has been reported to suppress innate immune responses through the targeting of ERK1/2 signaling and MTB Rv2387 appears to enable bacterial survival through TLR2/p38/JNK signaling. 201

| Vaccine relationship
The BCG vaccine for tuberculosis has been proposed and occasionally documented to effectively treat MAP-associated autoimmune diseases. 202BCG may have similar effects against CM.Vaccinations in general and the BCG vaccination in particular confer a lower risk for CM. 203In the case of vaccinations other than BCG, vaccination may act through the stimulation of heterologous or trained immunity against unrelated organisms. 202The BCG vaccine in contrast may act by the stimulation of immunity against another species of bovine mycobacteria.As previously mentioned, the BCG vaccine is an attenuated Mycobacterium bovis virus.[206] The successful treatment of CM with intralesional BCG further supports an association between MAP and CM.BCG is widely used throughout Asia as part of childhood immunization programs 207 and may underlie the low rates of CM in this population. 208lthough the BCG vaccine is not routinely administered in European countries such as the United Kingdom, it is occasionally used prophylactically to offset tuberculosis in high-risk individuals.However, it is not known if the rate of CM is lower among Britons who were vaccinated.

| ANIMAL MODELS
Animal models of melanoma using nude mice have focused almost exclusively on the possible role of UV radiation in melanoma development. 209,210We propose instead that dogs rather than mice will be excellent models of MAP-associated human CMs for the following reasons.
As discussed above, in countries where MAP infection of domestic livestock is longstanding, MAP is present throughout the soil of the country, in areas of both high and low density of cattle. 93The presence of MAP in soil may explain the development of CM in dogs, who may be infected with MAP from their near field exposure to MAP-contaminated soil.Most dog species bodies are covered by hair presumably minimizing the risk of melanoma related to UV exposure.Conversely, this coverage creates more opportunities for MAP to persist on canine hair and skin.
2][213] One review of animal models of human cancers highlights canine and humans' common environmental exposure to a "broad array of pathogens." 214

| LIMITATIONS AND STRENGTHS OF THE EVIDENCE
A recent review of the more well-known association of MAP with Crohn's disease discusses the many limitations in establishing MAP as the cause of human disease. 25Detecting the organism in human tissue is especially difficult owing to the variability in MAP detection methods and the putative existence of MAP in a cell wall deficient form that is not amenable to the usual mycobacterial stains.Systematic reviews of the detection of MAP in patients with Crohn's disease have generally supported a causative association. 24,215,216P has been identified in human blood, intestinal tissue, and lymph nodes, but not in human skin.Porcine skin 217,218 and human melanocyte cell lines 219 are available to test MAP's ability to penetrate skin and melanocytes, and the effect of MAP on these tissues and cells.Two studies have documented the presence of MAP on the surface of beef cattle hides but not within the epidermis. 220,221ile intriguing, the hypothesis that MAP exposures and CM are associated may not formally meet all nine criteria of Hill's correlates for causation (strength of association, consistency, specificity, temporality, biological gradient, plausibility, coherence, experiment, and analogy).However, these correlates are only guidelines.For example, A may cause B, yet may not necessarily meet all of Hill's criteria.Alternatively, a risk factor that meets all criteria may be determined to be a confounder rather than a true cause. 222,223ere is no research available that studies CM and immunosuppression involving MAP.Ideally this review will stimulate such research.As well, CM manifests significant intratumor and intertumoral heterogeneity, and varies by pathologic features (e.g., Breslow depth), transcriptomics, proteomics, and genomics. 224In the latter case, there are at least four genetic subtypes of melanoma (e.g., mutant BRAF, mutant RAS virus, mutant neurofibromatosis type 1 [NF1], and Triple-Wild-type) which may differentially interact with MAP. 225ile it may be argued that the current body of scientific literature does not provide adequate evidence of a direct link between MAP and melanoma, one is reminded of the now well-accepted causal association between Helicobacter pylori (H.pylori) and gastric cancer, which was similarly criticized for years as lacking consistent evidence.The current manuscript is the first time a possible causative association has been proposed between MAP and CM, and thus there are no epidemiologic studies or systematic reviews of this association.The more well known possible etiologic association between MAP and Crohn's disease has, in contrast, been the subject of numerous epidemiologic studies and systematic reviews.Despite the consistent evidence from these studies and reviews, MAP is still not generally accepted as a cause of Crohn's disease, highlighting the similar difficulty of establishing a link between MAP exposure and CM.
The relationship of CM with sun exposure is complicated and only partially explained by current scientific reasoning (e.g., intermittent exposure and dual pathway models). 226rguments contrary to a definitive cause and effect association for UV-radiation are multifold.This includes (1) a relatively small increase in incidence with age compared with squamous and basal cell skin cancers, (2) the historical predominance in women versus men, (3) an incongruence with areas of the body more versus less exposed to sunlight, (4) the comparatively rare occurrence of CM among black albinos, and (5) only marginally increased incidence with latitudes more proximal to the equator versus squamous and basal cell skin cancers. 227Accordingly, other nonsolar risks for CMs, such as MAP, are worth considering.
Exposure to MAP does not mean that one will develop melanoma, but rather that certain susceptible individuals may have an increased risk of developing the cancer, possibly years after the bacterium has been successfully eradicated from the body.Such is the case for H. pylori as a cause of gastric cancer, which has been demonstrated as long as 14 years after elimination of the bacterium. 228However, reinfection is a constant concern for both H. Pylori and MAP, requiring routine surveillance to assure the effectiveness of initial antibiotic therapy.Even then, primary and tertiary measures may only halve the cancer risk. 229Like H. Pylori infection and gastric cancer, only a fraction of MAP-exposed individuals potentially will manifest melanoma.
Since the hypothesis that MAP may cause some cases of human CM is new, there have been no efforts to identify MAP in human CM lesions.Of interest, Nobel laureate Zur Hausen discusses how the injection of the vaccinia virus into human skin has been associated with the subsequent development of CM at the injection site, and speculates that a "cattle virus" contaminating the vaccinia vaccine may be responsible for the CM. 58,230Mycobacterium leprae (ML) organisms have been identified in a basal cell carcinoma and a CM in a patient with ML, probably attributable to subsequent contamination of the already existing cancers with the ML organism. 231In the context of a previous discussion of the possible relationship between MAP and uveal melanoma, the ability of MAP to invade various human cell types, including enterocytes, macrophages, dendritic cells, and small intestinal goblet cells, was noted. 34ile genomic epidemiologic methods have advanced considerably in recent years for detecting MAP in isolates of ruminants, human detection still remains a challenge. 232The case in point being the lack of a gold standard such as culture upon which to validate results, appropriate guidance on best practices for selecting and handling human tissue specimens, and methods for the identification of referent groups by geography and prevalence of MAP in the environment are needed.The availability of targeted polymerase chain reaction (PCR) probes, or their mismatch, remains another concern given the genetic similarity of MAP with other mycobacteria of the Mycobacterium avium family. 233stablishing causality for a bacteria and cancer is difficult, especially when the determination of exposure is made at the point of diagnosis. 234Retrospective analyses are prone to the misclassification of cases as bacteria negative when individuals were infected in the past and the infection has since cleared.In lieu of costly randomized cohorts, nested case-referent studies present an alternative means of exploring the association between MAP and CM, although this design is subject to specimen degradation and differential attrition of the cohort over time.

| CONCLUSIONS
Melanoma skin cancer typically has been posited to result from exposure to sunlight as a source of UV radiation.This belief has limited the exploration of other hypothesized causes, especially where melanoma appears on the palms of the hands, soles of the feet, and on other areas of the body less exposed to the sun.The occurrence of primary gastric melanoma also raises questions about the exclusive role of sunlight in causing melanoma.These questions illustrate the need for further discussions on other possible causes of melanoma such as MAP.
When considered together, the evidence for MAP as a causative factor is not yet conclusive, with plausible alternative explanations.Future efforts will need to move beyond case examples that simply provide ecologic and patient characteristics at a single point in time.More epidemiologically rigorous and well-powered, population-based study designs are essential, as well as identifying best practices for the prevention and treatment of bacterial infections such as MAP.Lastly, individuals with MAP exposure may be susceptible to many different microbes having carcinogenic potential.Disentangling such etiologic agents will be key to determining if MAP is an independent risk factor for CM versus a coincidental risk.
In summary, the MAP hypothesis provides a new perspective on the pathogenic mechanisms underlying CM leading to better awareness of this disease.Ideally, the consideration of MAP as an etiologic agent may yield more effective prevention strategies and targeted treatment alternatives for CM.Mycobacterium avium subspecies paratuberculosis (MAP) exposure and related risk factors for cutaneous melanoma.Cutaneous melanoma is hypothesized to result from the interplay of MAP exposure, genetics, chemical exposures, and ultraviolet (UV) radiation.This combination leads to inflammation, oxidative stress, and infected melanocytes, setting the stage for cutaneous melanoma to develop.Potential alternative pathways for Mycobacterium avium subspecies paratuberculosis (MAP) exposure.Although MAP infection predominantly results from exposure to cattle manure where MAP is present, MAP infection can also occur from exposure to water or dirt contaminated by these bacteria.As illustrated above, both recreational and occupational activities can increase the risk of MAP exposure such as swimming, surfing, welding, cleaning chimney sweeps, and printing.The role of genetics in cutaneous melanoma development.Genetics have an indirect role in melanoma susceptibility, suggesting an interaction with environmental exposures (e.g., Mycobacterium avium subspecies paratuberculosis [MAP]).Many genes are independent of ultraviolet (UV) radiation such as phosphatase and tensin homolog (PTEN), breast cancer type 1 susceptibility protein (BRCA1), breast cancer type 2 susceptibility protein (BRCA2), retinoblastoma protein 1 (RB1), transformation-related protein 53 (TP53), BRCA1 associated protein 1 (BAP1), and glutamine synthetase gene-B (glnB).Typically, most variants associated with melanoma are somatic; however, a polygenic mode of inheritance involving the interplay of multiple low-risk alleles or rare mutations in high-penetrance genes such as adrenocortical dysplasia protein homolog (ACD), BAP1, cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase inhibitor 2A (CDKN2A/ p16INK4a), protection of telomeres 1 protein (POT1), telomeric repeat binding factor 2 interacting protein (TERF2IP), and telomerase reverse transcriptase (TERT) should not be ruled out.In non-pigment pathways, DNA repair, as suggested for melanocortin 1 receptors (MC1R), may underlie melanoma predisposition.Additionally, in vitro growth genes important to melanoma development include ribonucleic acid (RNA) polymerase sigma subfactor A (sigA), phosphoribosylaminoimidazole-succinocarboxamide synthase (PurC), phosphoribosylformylglycinamidine synthase subunit (PurL), and phosphoribosylglycinamide formyltransferase 1 (PurN).The significant genome-wide association with rs28986343 at the human leukocyte antigen (HLA) locus and the association between rs408825 and expression of the innate immunity gene interferoninduced guanosine triphosphate (GTP)-binding protein (MX2) supports an immunologic role for melanoma development.Immune system role in cutaneous melanoma risk and therapy.BCG vaccination potentially may act through the simulation of heterologous or trained immunity against Mycobacterium avium subspecies paratuberculosis (MAP).However, antigens vary among melanoma patients, presenting an obstacle to the development of individualized immune-related therapies.

FIGURE 2 .
FIGURE 2.Concentration of Mycobacterium avium subspecies paratuberculosis (MAP) associated with cattle manure.Emphasis is placed on the relationship between the amount of manure and doses of MAP.With more manure it is expected that increasing doses of MAP are present.

FIGURE 4 .
FIGURE 4.Importance of the gut-skin axis in the development of cutaneous melanoma.Patients with high levels of inflammatory cytokines, for example, interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-12 (IL-12), tumor necrosis factor (TNF), and interferon-γ (IFN-γ) may result from the "challenging to clear" Mycobacterium avium subspecies paratuberculosis (MAP) in the gut.This manifests as decreased integrity of the gut epithelium and microbiota imbalance (dysbiosis).Chronic inflammation, disruption of the gut-skin axis, and dysbiosis are important etiologic determinants of cutaneous melanoma risk.