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A hallmark feature of several pathogenic microbes is the distinctive color of their colonies when propagated in the clinical laboratory. Such pigmentation comes in a variety of hues, and has often proven useful in presumptive clinical diagnosis. Recent advances in microbial pigment biochemistry and the genetic basis of pigment production has sometimes revealed a more sinister aspect to these curious materials that change the color of reflected light by selective light absorbance. In many cases, the microbial pigment contributes to disease pathogenesis by interfering with host immune clearance mechanisms or by exhibiting pro-inflammatory or cytotoxic properties. Here, we review several examples of pigments that promote microbial virulence, including the golden staphyloxanthin of Staphylococcus aureus, the blue-green pyocyanin of Pseudomonas spp., and the dark brown or black melanin pigments of Cryptococcus neoformans and Aspergillus spp. Targeted pigment neutralization may represent a viable concept to enhance treatment of certain difficult infectious disease conditions.
Colors are vital to the sensing of the environment and have evolved in higher living organisms to guide their interactions with others. For example, it is well appreciated that many birds exhibit brightly colored plumage to attract members of the opposite sex, that a chameleon’s adaptation to surrounding color is an important means of camouflage, and that the bright coloration of the poison dart frog warn potential predators to stay away. But such explanations cannot be offered to explain why certain microorganisms are pigmented. Since they lack color perception, one must assume evolutionary selective pressures behind the acquisition of pigments that promotes survival independent of their light absorbance, reflection or emission spectral properties (Box 1).
These are some natural functions proposed for microbial pigments, with an example reference:
Because colors often provide an easy way of identifying certain microbes, they are often coined in names of species. For example, Rosenbach in 1884 named the golden-colored pathogen Staphylococcus aureus (aureus = “golden”, Latin) to distinguish it from nonpigmented staphylococci of the resident skin microflora that he named Staphylococcus alba (alba = “white”, Latin) 1. Likewise, the blue-green Pseudomonas species not infrequently found in the lungs of cystic fibrosis patients is given the name aeruginosa, which derives from a Latin word denoting the color of copper rust. Chromobacterium violaceum not surprisingly elaborates a blue-violet pigment. These hallmark phenotypes not only provide an easy nomenclature for the microorganisms, but continue to be important diagnostic clues in clinical laboratories today for the identification of microbes. Pigments have also played a role in the discovery of infectious pathogens. In the late 1870’s, while tending to pathology specimens from malaria patients in a military hospital in Algeria, Alphonse Laveron, a student of Pasteur, astutely noted that the only common element found in the blood and organs of these patients was a brown black pigment granule. This major observation was to open the gateway to discovery of the malaria parasite as the infecting agent for which Laveron was awarded a Nobel Prize in 19072.
With biotechnological advances, contemporary researchers are in position to study the molecular genetic and biochemical basis for microbial coloration. Investigations using purified pigments or isogenic mutants with altered pigmentation have begun to reveal how these molecules can provide a survival advantage for the pathogen in the host environment and/or produce significant alterations in host cells and immune response pathways (Table 1). In this article, we summarize our current understanding of microbial pigments and their possible role in the pathogenesis of human infectious disease.
Among the best-recognized bacterial pigments are the carotenoids that impart the eponymous golden color to the major human pathogen, Staphylococcus aureus. S. aureus produces multiple carotenoid pigments via a well-described biosynthetic pathway that culminates with golden staphyloxanthin (Figure 1a) as the major product and yellow 4′4′-diaponeurosporene as a minor product 3, 4. Deletion of the gene encoding the early staphyloxanthin biosynthesis enzyme CrtM renders the bacterium colorless and more susceptible to killing by human and mouse neutrophils or whole blood 5, 6. Loss of pigmentation translates to a significant decrease in S. aureus virulence in murine skin abscess or systemic infection models 5, 7. Interestingly, 4′4′-diaponeurosporene can be synthesized by a number of other bacteria upon transfer of just two S. aureus genes, crtM and crtN 3. When these genes are introduced into group A Streptococcus, the now pigmented tranformants produce large lesions in a mouse skin infection model, demonstrating S. aureus carotenoids are both necessary and sufficient to promote bacterial pathogenicity 5.
Staphyloxanthin consists of a C30 polyene carbon backbone with alternating single and double bonds typical of carotenoid pigments (Figure 1a); these alternating bonds are able to absorb excess energy from reactive oxygen species (ROS) 8. Compared to the wild-type parent strain, a nonpigmented S. aureus mutant is much more susceptible to killing by hydrogen peroxide, superoxide radical, hydroxyl radical, hypochloride and singlet oxygen 5, 6. Consistent with an antioxidant role, the survival advantage conferred by the S. aureus pigment is lost in killing assays using blood of chronic granulomatous disease (CGD) patients with deficient oxidative burst function, or upon infectious challenge of CGD mice 5.
Melanin is a pigment commonly found in organisms across many kingdoms (reviewed in 9–11). Melanins are structurally diverse high molecular pigments made of oxidative polymerization involving quinones, which can assume three oxidation states. Studies of the paramagnetic properties of melanin identified strong electron spin resonance signal, which is interpreted as evidence for the presence of stabilized free radicals in biological systems. Thus melanin can act as a trap for unpaired electrons and has the ability to stabilize potentially harmful unpaired electrons such as those from ROS 12. A normal component of our skin and hair, melanin is also found to coat the surface of two important fungal pathogens, Cryptococcus neoformans and Aspergillus fumigatus. Recent mutagenesis studies have confirmed a virulence function of melanin production of these two agents of severe opportunistic infection in immunocompromised patients.
C. neoformans is an encapsulated yeast-like fungus that produces a brown or black melanin pigment by conversion of diphenols or homogentisic acid 13 (Figure 1g); this coloration can be used for rapid identification of colonies on cornmeal agar, a medium commonly used in yeast isolation 14. C. neoformans deficient in melanin production are less invasive and survive poorly in the spleen, liver or brain of infected animals 15, 16. This diminished virulence correlates to a reduced ability of melanin-deficient mutants to resist phagocytic killing in vitro 17. Melanin production impedes phagocytosis of encapsulated C. neoformans by macrophages in vitro and in a murine lung infection model 17, 18. Melanin also interferes with the action and efficacy of endogenous antimicrobial peptides and pharmacologic antifungal agents against C. neoformans. The negatively charged pigment neutralized the activity of neutrophil defensin and other cationic antimicrobial peptides 19, and bound avidly to amphotericin B and caspofungin 20, two front line drugs used in the treatment of severe fungal infection. Lastly, a ferric iron reduction property of C. neoformans melanin, converting Fe3+ to Fe2+, could theoretically facilitate ferrous iron through a specific transport system and improve in vivo survival 21, 22.
Melanin pigment production may also modulate the inflammatory response to cryptococcal infection. When compared to a weakly pigmented strain, infection with a heavily melanized strain of C. neoformans inhibited the afferent phase of the T-cell immune response as evidenced by diminished TNF-α production by alveolar macrophages and decreased expansion of cryptococcus-specific lymphocytes 23. Further evidence of pigment mediated inflammatory gene suppression comes from analysis of central nervous system (CNS) injury and cytokine responses following direct intracerebral instillation of an albino C. neoformans strain versus a companion melanotic revertant. The pigmented strain produced a lethal infection and massive CNS tissue damage accompanied by minimal cytokine response. Conversely, the melanin-deficient strain never produced a fatal infection, and triggered enhanced CNS levels of mRNA transcripts for IL-12, TNF-α, IL-1β, INF-γ and iNOS 24. Mouse immunization studies using cryptococcal melanin have shown that, despite its amorphic polymeric nature, the fungal pigment can stimulate the immune system to generate specific antibodies 25.
A. fumigatus is a filamentous fungus that elaborates a melanin-like substance during its conidial stage of growth. Survival of conidia within the host is a critical first step in Aspergillus infection. Conidia from an A. fumigatus mutant strain lacking pigmentation are more susceptible to killing by oxidants and by human monocytes in vitro, and showed reduced virulence in a murine infectious challenge model 26. Electron microscopic analysis demonstrated that nonpigmented conidia sustained more extensive structural damage within monocytes compared to wild-type pigmented conidia 26. Targeted mutation of the A. fumigatus alb1 gene, encoding a polyketide synthase in the dihydroxynaphthalene-melanin pathway, results in an albino phenotype lacking the bluish-green conidial pigment 27. The nonpigmented mutant was found to be much more susceptible to complement C3 deposition, neutrophil phagocytosis, and was significantly attenuated in a murine intravenous challenge model 28.
Additional evidence for a contribution of melanin pigments to virulence has been provided in studies of other fungal and bacterial pathogens. Elimination of melanin production by the infrequently encountered dematiaceous fungus Wangiella dermatitidis is associated with diminished ability to produce invasive hyphal forms, increased susceptibility to neutrophil killing, and virulence in mouse models of infection 29, 30. Non-melanized conidial mutants of the thermally-dimorphic fungal pathogen Sporothrix schenckii show increased susceptibility to killing by ROS, reactive nitrogen species or UV light 31. Proteus mirabilis, a Gram-negative bacterial agent of human urinary tract infections, produces a melanin pigment that can act as a free radical trap 32. A melanin pigment isolated from an epidemic strain of Burkholderia cepacia also possesses antioxidant properties that can attenuate macrophage superoxide production 33.
P. aeruginosa is a leading bacterial pathogen in hospital settings and patients that are immuno-compromised due to neutropenia, burns or cystic fibrosis. Many P. aeruginosa strains elaborate the blue-green phenazine-derived pigment pyocyanin (Figure 1e), which can impart a greenish hue to the sputum of cystic fibrosis patients with chronic lung infection 34. In contrast to the antioxidant features of staphyloxanthin and bacterial melanin pigments, P. aeruginosa pyocyanin exhibits a paradoxical pro-oxidant property. A zwitterion that can easily penetrate biological membranes, pyocyanin can directly accept electrons from reducing agents such as NADPH and reduced glutathione, then transfer the electrons to oxygen to generate ROS such as hydrogen peroxide and singlet oxygen 35 at the expense of host antioxidant systems such as glutathione and catalase 36. P. aeruginosa mutants lacking pyocyanin are greatly attenuated in both acute and chronic mouse models of lung infection 37, and the remarkable toxic properties of the pigment can be demonstrated to extend to a broad array of target organisms including bacteria, yeast, insects, nematodes, and plants 37–40. Inhibition of cellular respiration is clearly one of the important mechanisms of pyocyanin toxicity to bacterial or eukaryotic cells 41, 42.
The fundamental ability of pyocyanin to alter the redox cycle and increase oxidative stress appears central to its diverse detrimental effects on host cells. For example, pyocyanin disrupts Ca2+ homeostasis in human airway epithelial cells by oxidant-dependent increases in inositol trisphosphate and the abnormal release of Ca2+ from intracellular stores. Since Ca2+ is important for regulating ion transport, mucus secretion and ciliary beat, these alterations likely have important ramification for P. aeruginosa lung infections 43. The pathway of vacuolar ATPase vesicle transport and protein targeting appears particularly sensitive to pyocyanin action, as revealed in a yeast mutant library screen 42. Pyocyanin inhibition of ATPase could directly explain many of its toxicities including ciliary dysmotility 44, disruption of calcium homeostasis 43, and diminished apical membrane localization of the cystic fibrosis transmembrane conductance regulator (CFTR) 45. Other potentially toxic effects of pyocyanin include perturbance of cellular respiration, epidermal cell growth inhibition, prostacyclin release from lung endothelial cells, and altered balance of protease-antiprotease activity in the cystic fibrosis lung 46.
Many ROS exert a direct impact on NF-kB and other signaling pathways to boost inflammatory cytokine secretion 47, 48. The prooxidant effect of pyocyanin can thus augment such innate immune response circuits 49. For example, pyocyanin increases the release of neutrophil chemokine IL-8 from lung epithelial cells and upregulates the expression of the neutrophil receptor intracellular adhesion molecule-1 (ICAM-1) both in vitro and in vivo; these proinflammatory effects were blocked by treatment with antioxidants 49, 50. In neutrophils, pyocyanin induces a sustained increase in ROS and subsequent decrease in intracellular cAMP, which triggers a time- and concentration-dependent acceleration of apoptosis 51. As confirmed in studies using WT and isogenic pyocyanin-deficient mutant P. aeruginosa, pigment-dependent acceleration of neutrophil apoptosis and diminished release of neutrophil chemokines may represent an immune suppression mechanism of the pathogen 52.
Malaria parasites, including the human pathogens Plasmodium falciparum, P. vivax, P. malariae, and P. ovale, accumulate a brown pigment during infection known as hemozoin 53. In its strictest sense the pigment is not a plasmodial product, but rather the byproduct of heme detoxification 54 (Figure 1b). There exist many theories as to how hemozoin is made, be it by some host processes or specific plasmodial detoxification enzyme 55. Hemozoin has many functions that could contribute to Plasmodium virulence, and importantly, several antimalarial drugs including chloroquine work by targeting this heme detoxification/hemozoin synthesis pathway.
The hemozoin pigment appears to exert mixed effects on the host immune system. Ingestion of hemozoin released during schizont rupture by phagocytes has been shown to lead to depression of phagocytosis and oxidative burst, likely due to iron intoxication as removal of the labile iron fraction from pigment reduces pigment toxicity 56. Hemozoin, and/or products bound by the pigment, also decrease expression of MHC class II antigen, CD54, and CD11c in human monocytes thereby impacting antigen presentation 57 and blocking differentiation and maturation of human monocyte-derived dendritic cells 58. Conversely, purified hemozoin activates macrophages to produce pro-inflammatory cytokines, chemokines, and nitric oxide 59, which together are thought to contribute to many of the systemic symptoms of malaria. Initial study of this phenomenon linked hemozoin activity to the TLR9 immune activation pathway 60. Subsequent work has shown that hemozoin itself is inert as nuclease treatment abolished proinflammatory functions, indicating that the pigment serves as a carrier for plasmodial DNA which itself is important in activating the host cytokine response 61.
Group B Streptococcus (GBS), the leading etiologic cause of severe neonatal bacterial infection, expresses an orange-red pigment that was initially thought to be a carotenoid due to its signature triple peak absorbance pattern 62, 63. However a more recent report deduced the pigment structure to be an ornithine rhamno-polyene with 12 conjugated double bonds, dubbed granadaene 64 (Figure 1d). GBS pigment has been shown to enhance GBS survival within macrophages 65, and study of isogenic pigmented versus nonpigmented GBS showed preferential survival of the pigmented GBS in systemic infection models 65. Expression of the pigment is invariably linked to expression of another well-known GBS virulence factor, the pore-forming β-hemolysin/cytolysin, through a single genetic locus known as the cyl operon 66, 67.
Violacein is a deep violet pigment produced by Chromobacterium violaceum, an occasional agent of fatal septicemia in humans 68. Oxidation and coupling of two molecules of L-tryptophan by the VioA to VioE enzymes generate the rearranged pyrrolidone-containing scaffold of the final pigment 69 (Figure 1c). Violacein has been demonstrated to possess strong antioxidant properties, and can protect lipid membranes from peroxidation caused by hydroxyl radicals 70. Investigation of violacein as a chemotherapeutic agent reveal its capacity to induce apoptosis of leukocyte cell lines 71, and it is conceivable this property could play a role in immune evasion during severe human infections. Finally, violacein has potent antimicrobial activity against many bacteria and protozoa 68, 72. Hence, secretion of this pigment may protect against protozoal predation 73 and promote survival of C. violaceum in the environment.
The Gram-negative rod-shaped anaerobic bacterium Porphyromonas gingivalis is implicated in the pathogenesis of certain forms of periodontal disease. Arginine- and lysine-specific gingipain proteases of P. gingivalis degrade hemoglobin to release iron(III) protoporphyrin IX (Figure 1b), which is dimerized to form the micro-oxo bis-haem-containing black pigment of the organism 74. This pigment can then act as a buffer for P. gingivalis against killing by ROS generated by neutrophils 75.
Since information is available on the biosynthetic pathways underlying pigment generation in several pathogenic species, the pigments themselves become logical targets for virulence factor-based therapeutic interventions. For example, the first committed step in staphyloxanthin biosynthesis, catalyzed by the CrtM enzyme, involves the head-to-head condensation of two molecules of farnesyl diphosphate to produce the C30 species, presqualene diphosphate 6. This reaction resembled a key step used in human cholesterol biosynthesis, catalyzed by squalene synthetase (SQS). Solution of the S. aureus CrtM crystal structure revealed active site similarities, and it was found that several SQS inhibitors developed in the context of cholesterol lowing activity also inhibited staphylococcal pigmentation 7. One such inhibitor, a phosphonosulfonate, was shown effective in rendering S. aureus susceptible to ROS and neutrophil killing, and was effective at reducing levels of the pathogen by 98% in a murine systemic infection model 7. Theoretical advantages of this therapeutic approach would lie in specificity, since the drug would not exert unwanted effects on the normal microflora, and reduced selective pressure for resistance, since the drug only exerts its killing effect in the disease context of an activated host immune response 5, 7
Novel approaches to treatment of cryptococcal infection by inhibition of melanin production have been explored. The systemic herbicide glyphosphate depletes C. neoformans melanin levels, and prolongs host survival in an experimental mouse model of cryptococcosis 76. Therapy of C. neoformans-infected mice with monoclonal antibodies to melanin reduced fungal burden 100-fold and improved survival following lethal challenge 77. Since melanin also binds to amphotericin B and caspofungin, synergistic use of a melanin inhibitor could further improve efficacy of these major antifungal drugs 20.
The reddish-pink linear trypyrrole pigment prodigiosin (Figure 1f) is produced by Serratia marcesens, an agent of nosocomial infections of the urinary tract and wounds. Prodigiosin has cytotoxic activity against numerous cancer cell lines 78, 79 and an immunosuppressive effect on T cells, blocking IL-2 dependent proliferation through inhibition of IL-2-Rα expression 80. In animal studies, prodigiosin blocks tumor metastasis, delays onset of autoimmune diabetes and arthritis, and improves survival in heart transplant and graft-versus-host disease 78, 79. Violacein extracted from C. violaceum is effective against multiple cancer cells including uveal melanoma, colorectal, and leukemia, lymphoma cells in culture 71, 81, 82
Synthetic melanin and melanin derived from grapes have been shown to downregulate pro-inflammatory cytokine production in the presence of human blood monocytes and in a rat model of adjuvant induced inflammatory disease respectively. 83, 84. Likewise, a few carotenoids have been shown to activate the steroid receptor RAR and RXR pathways to directly contribute to immune suppression 85. Whether melanins and carotenoids isolated from microbes have immunosuppressive properties remains to be discovered.
Finally, to engineer natural products most suitable for human consumption, researchers have begun to develop recombinant microorganisms through engineering novel biosynthetic pathways by (a) the combination of compatible genes from different genomes into functional clusters, and (b) the further evolution of new enzyme functions of these genes via experimental mutagenesis, recombination and selection 86, 87.
Color in many animals warns of impending danger. From the evidence summarized in this review, it would not be too farfetched to say that pigmentation elaborated by certain microbial species provides a warning of enhanced pathogenic potential. While phylogenetic diversity of pigmented microbial species and the chemical diversity of the pigments themselves may preclude a single unifying hypothesis for their evolution and persistence, the most common virulence-associated theme identified among microbial pigments is resistance against ROS. The ability of many pigments to stabilize ROS may be inherently linked to the ability of these compounds to confer color sensorium. We postulate that most pigments evolved initially as a mechanism to combat environmental ROS, but over time, these compounds were adapted to serve divergent functions.
Pigmentation may contribute to virulence by allowing a given microbe to evade host immune killing or by provoking inflammatory damage to cells and tissues. The danger of pigmented pathogens may be further heightened in patients with particular immunodeficiencies. For example, patients with CGD harbor mutations in NADPH oxidase resulting in weak phagocyte oxidative burst function; these individuals suffer chronic deep-seated infections with several pigmented microorganisms such as S. aureus, Aspergillus spp., S. marcescens, and B. cepacia atop the list of etiologic agents 88, perhaps due to effective neutralization of all residual ROS.
Further understanding of the biological properties of microbial pigments will not only enrich our instinctual curiosity about colors, but also provide a scientific basis for therapeutic disarming of the pathogens or for borrowing these multifunctional molecules in pharmacologic applications (Box 2).
Our research on S. aureus and GBS pigments was supported by NIH grants AIO7432 (GYL) and HD051796 (VN), a Burroughs–Wellcome Career Award (GYL), and an American Heart Association Established Investigator Award (VN).
The authors are each on the scientific advisory board of the biotechnology company Auricx Pharmaceuticals, Inc.
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