Secondary metabolites from bacteria inhabiting extreme anthropogenic environments

Secondary metabolites, also known as specialized metabolites, are molecules that are not essential for the survival of bacteria under laboratory conditions but likely provide significant benefits to the producer in natural environments. These metabolites are produced by soil bacteria belonging to the phyla Actinomycetota (actinomycetes), Bacillota (bacilli), and Myxococcota (myxobacteria). Soil represents a complex environment where various types of bacteria compete for nutrient sources, possessing vast arsenals of secondary metabolites for a range of interactions.

The chemical and physiological functions of secondary metabolites are highly diverse, and they play roles in various aspects of human life, including as antibiotics, pesticides, antiparasitic drugs, herbicides, anti-inflammatory drugs, cardioactive compounds, antitumor drugs, antiviral drugs, antioxidants, and immunoactive modulators and stimulators. These bioactivities often overlap: more than half of the known secondary metabolites also exhibit other bioactivities. Undoubtedly, the application of bacterial secondary metabolites has revolutionized fields such as medicine, agriculture, and biotechnology.

Actinomycetes

Actinomycetes, especially those within the genus Streptomyces, are unparalleled in their ability to produce antibiotics. This group is responsible for the discovery of over two-thirds of naturally derived antibiotics which are staples in clinical treatments against bacterial infections. Actinomycetes inhabit soil and decomposing organic matter, environments ripe with microbial competition, prompting these organisms to evolve chemical defenses in the form of secondary metabolites. Their metabolic repertoire extends beyond antibiotics to include antifungals, antiparasitic agents, immunosuppressants, and anticancer compounds, making them a cornerstone in the development of new pharmaceuticals.

Bacilli

Bacilli are notable for their diverse production of bioactive molecules, with Bacillus subtilis and related species being particularly significant. These bacteria are known for synthesizing lipopeptides like surfactin, which possess antibiotic properties and are utilized in bioremediation and as industrial biosurfactants. Bacilli also produce polymyxins, critical for treating infections caused by multi-drug resistant bacteria, underscoring their role in combating antibiotic resistance. Furthermore, bacilli are a source of industrially relevant enzymes and biopesticides, demonstrating their versatility and utility beyond pharmaceutical applications. Last bot not the least, members of Bacillota phylum carry genes for an untapped variety of CRISPR-associated enzymes (e.g. Cas9).

Myxobacteria

Myxobacteria exhibit complex social behaviors and are distinguished by their ability to produce secondary metabolites with unique and potent biological activities. These soil-dwelling organisms have been a source of novel antibacterial, antifungal, and anticancer agents. Notably, compounds like epothilones from myxobacteria have shown promising anticancer properties in clinical trials, reflecting their potential in developing new therapeutic strategies against cancer. Myxobacteria's unique life cycle and social interactions foster the evolution of distinctive biosynthetic pathways, contributing to the structural and functional diversity of their natural products.

Key components of novel secondary metabolites discovery pipeline

Isolation of bacteria from natural environments.

Samples of water, soil, other substrates are collected in order to isolate bacteria. Cultivable bacteria are the most important source of novel secondary metabolites. Each group of bacteria has defined conditions for the isolation and laboratory cultivation, allowing to isolate specific bacteria.

Isolation of bacteria from natural environments.

Biosynthetic gene clusters for secondary metabolites

Secondary metabolites are complex organic compounds synthesized via cascades of enzymatic reactions inside bacterial cell. One of the most important discoveries of last century is understanding, that enzymes for the biosynthesis of bacterial secondary metabolites are encoded within so-called biosynthetic gene clusters. These are assemblies of genes, which are i) co-localized on a chromosome; ii) co-expressed; and iii) co-regulated. Advanced tools of computer analysis allow to scan bacterial genomes in order to identify biosynthetic gene clusters and predict ability to produce secondary metabolites

Antimicrobials from bacterial sources

Antibiotics have longer history then one might expect. Synthetic antibiotic-like molecules appeared in Europe in the beginning of 20th century. However, human imagination is limited; real boom of antibiotics started when antimicrobial compounds from natural sources were discovered. Everyone knows about the discovery of penicillin by Alexander Flemming, however “golden age” of antibiotics started when Selman Waksman pointed on actinomycetes – mycelium-forming soil-dwelling Gram-positive bacteria – as on virtually infinite source of antibiotics.

Selman Waksman proposed the term “antibiotic”, to describe chemical compounds produced by microorganisms able to inhibit or terminate growth of bacteria. Nowadays, the term has broader usage, including synthetic (possibly mimicking natural) and semisynthetic compounds. Moreover, term antibiotic is often used to describe secondary metabolites active against fungi, protists, or even multicellular eukaryotes. However, in strict sense antibiotics are antimicrobial compounds.

Main targets of antibiotics.

As antibiotics are used to treat bacterial pathogens in humans (or animals), they should target parts of the bacterial cell that are absent in eukaryotic cells. Otherwise, antibiotics could also harm the cells of the treated organism, which must be avoided. What are these main targets?

1) Cell wall Biosynthesis.

The bacterial cell wall is a rigid, mesh-like structure composed of peptidoglycan molecules. It is vital for maintaining cell shape and integrity, provides turgor, serves as a protective shell, and acts as a semi-permeable barrier. Animal cells lack a cell wall, making it an ideal target for antibiotics. Furthermore, the biosynthesis of peptidoglycan and cell wall assembly is a complex process involving numerous enzymes and precursor molecules. With so many targets, multiple groups of antibiotics can terminate or inhibit cell wall biosynthesis in Gram-positive or Gram-negative bacteria. Currently, there are two groups of antibiotics targeting cell wall biosynthesis that are used in humans: β-lactam and glycopeptide antibiotics. The first group is best known for its founding member, penicillin, although hundreds of natural and semi-synthetic β-lactams are known today, with many used in clinical practice. Meanwhile, the best-known glycopeptides are vancomycin and teicoplanin, which are used to treat serious infections in both infants and adults.

2) Protein biosynthesis (translation).

The bacterial ribosome has a different structure than the ribosome of eukaryotes (including humans and other animals). Consequently, protein biosynthesis in bacteria serves as another excellent target for antibiotics. Clinically-used antibiotics that target protein biosynthesis in bacteria include aminoglycosides, tetracyclines, amphenicols, macrolides, and lincosamides.

3) mRNA biosynthesis (transcription).

Bacterial RNA polymerase also has a different structure from that of eukaryotes, offering yet another target for antibiotics. Rifamycins are antibiotics that bind bacterial RNA polymerase and block transcription. Rifamycins are active against various bacteria and are most importantly used to treat tuberculosis.

• Aminoglycosides are most effective against aerobic Gram-negative bacteria and are ineffective against Gram-negative anaerobes. Often, aminoglycosides are combined with β-lactams.

• Tetracyclines are broad-spectrum antibiotics, active against various Gram-positive and Gram-negative bacteria. Moreover, tetracyclines are among the most affordable antibiotics, allowing for broad application.

• Amphenicols are broad-spectrum antibiotics as well. Initially, they were used to treat typhoid; however, currently, amphenicols are being reconsidered as a treatment for other resistant bacteria.

• Macrolides are primarily used to treat infections caused by Gram-positive pathogens; they are also affordable and widely utilized.

• Lincosamides are active against Gram-positive bacteria, while Gram-negatives are inherently resistant to lincosamides. These antibiotics are often used in clinical practice for patients allergic to β-lactams.

ESKAPE

ESKAPE pathogens are a group of bacteria known for their ability to "escape" the effects of antibiotics, making them highly significant in the field of hospital-acquired infections. This group includes six notorious pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. They are particularly dangerous because they have developed strong resistance mechanisms against most available antimicrobial agents, posing serious treatment challenges. Consequently, ESKAPE pathogens are a focus of current research efforts aimed at developing new and effective antimicrobial strategies to combat these resistant bacteria and reduce their impact on public health

Antifungal secondary metabolites from bacteria

Different species of fungi can cause acute or opportunistic infections in humans. Fungal infections are often difficult to diagnose due to their subtle manifestations in the early stages, while treatment at later stages requires the use of various classes of antifungal compounds.
The leading class of antifungal compounds is the polyenes, secondary metabolites produced by actinomycetes. The founding member of the polyene antifungals, amphotericin B, was isolated from Streptomyces nodosus in 1955, entered clinical use in 1958, and remains widely utilized. Amphotericin B and other polyenes bind to ergosterol, an integral component of the fungal cell membrane. This binding induces pore formation, leading to ion leakage and subsequent cell death.
Resistance to polyenes is relatively rare; however, the spread of resistant Aspergillus and Candida spp. has increased in recent years. Especially alarming is the spread of multidrug resistant strains of Candida auris. Fortunately, actinomycetes, especially Streptomyces spp., are prolific sources of polyenes. The continuous search for novel polyene antifungals is essential for tackling fungal resistance.

Most serious human pathogen of fungal origin

Aspergillus fumigatus

Aspergillus fumigatus, is a mold found in soil and decaying organic matter. It is the primary cause of aspergillosis, a spectrum of diseases affecting the respiratory system. This fungus is most notable for causing invasive pulmonary aspergillosis in immunocompromised patients, such as those undergoing hematopoietic stem cell or solid organ transplantation, or receiving immunosuppressive therapy. Inhalation of airborne conidia (spores) can lead to colonization and infection of the lung tissues, presenting clinically as persistent cough, fever, chest pain, and occasionally hemoptysis. The pathology often involves angioinvasion, where the fungus invades blood vessels, leading to thrombosis and infarction of lung tissue. Diagnostic approaches include imaging, notably chest CT scans showing characteristic 'halo' signs, direct microscopic examination of sputum, and culture of the organism. Treatment typically involves antifungal agents like voriconazole or amphotericin B. Given the high mortality associated with invasive aspergillosis, early diagnosis and treatment are crucial.

Aspergillus fumigatus

Anticancer compounds from bacterial sources

Bacteria are able to produce different secondary metabolites that are cytotoxic. Term “cytotoxic” means that a certain compound is able to kill eukaryotic cells in vitro and in vivo, inducing either necrosis or apoptosis. However, it is important that certain compounds should have higher affinity to cancer cells, also having antiproliferative (i.e. suppressing the growth of malignant cells into surrounding tissue) and antineoplastic (i.e. suppressing or preventing the formation of tumor) properties in vivo.

Several groups of bacterial secondary metabolites have demonstrated such properties and are investigated as a promising treatment options for different types of cancer. However, as of today, only one group of such compounds are actively used in clinics. These are antracyclines, actinomycin, and bleomycin. All these compounds are able to interfere with DNA, affecting rapidly growing cells and hence having higher affinity for actively dividing malignant cells.

1) Anthracyclines

Anthracyclines are a class of potent anticancer drugs widely used in chemotherapy regimens for a variety of cancers, including leukemias, lymphomas, breast cancer, and sarcomas. Anthracyclines are produced by different Streptomyces spp., and the most notable members of this class include doxorubicin, daunorubicin, idarubicin, and epirubicin. These compounds are known for their ability to intercalate into DNA, disrupting the function of topoisomerase II, an enzyme crucial for DNA replication and transcription. This disruption leads to the inhibition of DNA synthesis and the induction of apoptosis in cancer cells.

Anthracyclines are also known for generating free radicals, which contribute further to their cytotoxic effects by damaging cellular membranes, DNA, and proteins. However, their clinical use is often limited by a significant side effect: cardiotoxicity, which can lead to long-term heart damage. Despite this, anthracyclines remain a cornerstone of cancer therapy due to their efficacy, and ongoing research aims to develop derivatives with reduced cardiotoxic effects and improved cancer-targeting abilities.

2) Actinomycin D, also known as dactinomycin

Actinomycin D, also known as dactinomycin, is a chemotherapy drug used primarily in the treatment of various cancers, including Wilms' tumor, rhabdomyosarcoma, Ewing's sarcoma, and certain types of ovarian and testicular cancers. This compound is derived from the bacterium Streptomyces parvulus and was one of the first secondary metabolites shown to have anticancer activity.

Actinomycin D exerts its anticancer effects by intercalating into DNA, where it preferentially binds to guanine residues and inhibits the transcription process by blocking the elongation phase by RNA polymerase. This inhibition prevents the synthesis of mRNA and subsequently protein synthesis, leading to cell death.
Despite its effectiveness, the use of actinomycin D is limited by its toxicity, which can include severe effects on the gastrointestinal tract, bone marrow suppression, and effects on the liver and kidneys. Its use is typically reserved for specific types of cancer where it has been shown to improve survival outcomes, often as part of a multi-drug chemotherapy regimen.

3) Bleomycins

Bleomycins serve as effective anticancer agents. These drugs are primarily used to treat Hodgkin's lymphoma, non-Hodgkin's lymphoma, testicular cancer, and certain types of head and neck cancers. Bleomycins are derived from the bacterium Streptomyces verticillus and bind to DNA causing strand breaks through oxidative damage. This leads to cell cycle arrest and apoptosis in cancer cells.

Bleomycins are administered via intramuscular, intravenous, or subcutaneous routes, allowing for flexible dosing in clinical settings. One of the notable advantages of bleomycins is their minimal myelosuppressive effects, making them a preferred option in combination chemotherapy regimens where bone marrow preservation is crucial.
However, bleomycin usage is significantly limited by its potential to cause pulmonary toxicity, which can manifest as pulmonary fibrosis, particularly with higher cumulative doses. This side effect necessitates careful monitoring of lung function during treatment.