Characterization of the skin microbiome in normal and cutaneous squamous cell carcinoma affected cats and dogs.

Human cutaneous squamous cell carcinomas (SCCs) and actinic keratoses (AK) display microbial dysbiosis with an enrichment of staphylococcal species, which have been implicated in AK and SCC progression. SCCs are common in both felines and canines and are often diagnosed at late stages leading to high disease morbidity and mortality rates. Although recent studies support the involvement of the skin microbiome in AK and SCC progression in humans, there is no knowledge of this in companion animals. Here, we provide microbiome data for SCC in cats and dogs using culture-independent molecular profiling and show a significant decrease in microbial alpha diversity on SCC lesions compared to normal skin (P ≤ 0.05). Similar to human skin cancer, SCC samples had an elevated abundance of staphylococci relative to normal skin-50% (6/12) had >50% staphylococci, as did 16% (4/25) of perilesional samples. Analysis of Staphylococcus at the species level revealed an enrichment of the pathogenic species Staphylococcus felis in cat SCC samples, a higher prevalence of Staphylococcus pseudintermedius in dogs, and a higher abundance of Staphylococcus aureus compared to normal skin in both companion animals. Additionally, a comparison of previously published human SCC and perilesional samples against the present pet samples revealed that Staphylococcus was the most prevalent genera across human and companion animals for both sample types. Similarities between the microbial profile of human and cat/dog SCC lesions should facilitate future skin cancer research. IMPORTANCE: The progression of precancerous actinic keratosis lesions (AK) to cutaneous squamous cell carcinoma (SCC) is poorly understood in humans and companion animals, despite causing a significant burden of disease. Recent studies have revealed that the microbiota may play a significant role in disease progression. Staphylococcus aureus has been found in high abundance on AK and SCC lesions, where it secretes DNA-damaging toxins, which could potentiate tumorigenesis. Currently, a suitable animal model to investigate this relationship is lacking. Thus, we examined the microbiome of cutaneous SCC in pets, revealing similarities to humans, with increased staphylococci and reduced commensals on SCC lesions and peri-lesional skin compared to normal skin. Two genera that were in abundance in SCC samples have also been found in human oral SCC lesions. These findings suggest the potential suitability of pets as a model for studying microbiome-related skin cancer progression.

An antibiotic from an uncultured bacterium binds to an immutable target.

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, lipid II, and lipid IIIWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.

A new antibiotic from an uncultured bacterium binds to an immutable target.

Antimicrobial resistance is a leading mortality factor worldwide. Here we report the discovery of clovibactin, a new antibiotic, isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant bacterial pathogens without detectable resistance. Using biochemical assays, solid-state NMR, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C 55 PP, Lipid II, Lipid WTA ). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate, but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the irreversible sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. Uncultured bacteria offer a rich reservoir of antibiotics with new mechanisms of action that could replenish the antimicrobial discovery pipeline.

Skin Cancer-Associated S. aureus Strains Can Induce DNA Damage in Human Keratinocytes by Downregulating DNA Repair and Promoting Oxidative Stress.

Actinic keratosis (AK) is a premalignant lesion, common on severely photodamaged skin, that can progress over time to cutaneous squamous cell carcinoma (SCC). A high bacterial load of Staphylococcus aureus is associated with AK and SCC, but it is unknown whether this has a direct impact on skin cancer development. To determine whether S. aureus can have cancer-promoting effects on skin cells, we performed RNA sequencing and shotgun proteomics on primary human keratinocytes after challenge with sterile culture supernatant ('secretome') from four S. aureus clinical strains isolated from AK and SCC. Secretomes of two of the S. aureus strains induced keratinocytes to overexpress biomarkers associated with skin carcinogenesis and upregulated the expression of enzymes linked to reduced skin barrier function. Further, these strains induced oxidative stress markers and all secretomes downregulated DNA repair mechanisms. Subsequent experiments on an expanded set of lesion-associated S. aureus strains confirmed that exposure to their secretomes led to increased oxidative stress and DNA damage in primary human keratinocytes. A significant correlation between the concentration of S. aureus phenol soluble modulin toxins in secretome and the secretome-induced level of oxidative stress and genotoxicity in keratinocytes was observed. Taken together, these data demonstrate that secreted compounds from lesion-associated clinical isolates of S. aureus can have cancer-promoting effects in keratinocytes that may be relevant to skin oncogenesis.

Changes in the skin microbiome associated with squamous cell carcinoma in transplant recipients.

Actinic keratoses (AK) arise in severely photo-damaged skin and can progress to squamous cell carcinomas (SCC). AK and SCC are common in Caucasian populations, and immunosuppressed individuals have a markedly higher risk of developing SCC. An overabundance of Staphylococcus aureus has been reported in AK and SCC lesions of immunocompetent individuals, however, the AK/SCC microbiome in immunosuppressed cohorts has not been investigated. Here, the microbial profile and bacterial load of AK, SCC and control skin swabs from 32 immunosuppressed organ transplant recipients were characterised via SSU rRNA gene sequencing and qPCR, and compared to a previously described immunocompetent cohort. Although the taxonomic composition of skin swab samples was mostly subject-specific, significant differences were observed between control skin, AK, and SCC in both cohorts. Surface bacterial load was increased and alpha diversity decreased in AK and SCC compared to control skin due to an increased abundance of Staphylococcus species and relative decrease of skin commensals. Staphylococcus epidermidis predominated on SCC from transplant recipients in contrast to SCC of immunocompetent subjects dominated by S. aureus. In conclusion, AK and SCC of immunosuppressed and immunocompetent subjects present with distinctive microbial dysbioses, which may be relevant to SCC pathogenesis and progression.

Secreted Toxins From Staphylococcus aureus Strains Isolated From Keratinocyte Skin Cancers Mediate Pro-tumorigenic Inflammatory Responses in the Skin.

Squamous cell carcinoma (SCC) is a common type of skin cancer that typically arises from premalignant precursor lesions named actinic keratoses (AK). Chronic inflammation is a well-known promoter of skin cancer progression. AK and SCC have been associated with an overabundance of the bacterium Staphylococcus aureus (S. aureus). Certain secreted products from S. aureus are known to promote cutaneous pro-inflammatory responses; however, not all S. aureus strains produce these. As inflammation plays a key role in SCC development, we investigated the pro-inflammatory potential and toxin secretion profiles of skin-cancer associated S. aureus. Sterile culture supernatants ("secretomes") of S. aureus clinical strains isolated from AK and SCC were applied to human keratinocytes in vitro. Some S. aureus secretomes induced keratinocytes to overexpress inflammatory mediators that have been linked to skin carcinogenesis, including IL-6, IL-8, and TNFα. A large phenotypic variation between the tested clinical strains was observed. Strains that are highly pro-inflammatory in vitro also caused more pronounced skin inflammation in mice. Proteomic characterization of S. aureus secretomes using mass spectrometry established that specific S. aureus enzymes and cytolytic toxins, including hemolysins, phenol-soluble modulins, and serine proteases, as well as currently uncharacterized proteins, correlate with the pro-inflammatory S. aureus phenotype. This study is the first to describe the toxin secretion profiles of AK and SCC-associated S. aureus, and their potential to induce a pro-inflammatory environment in the skin. Further studies are needed to establish whether these S. aureus products promote SCC development by mediating chronic inflammation.

Antibody-Free Multiplex Measurement of 23 Human Cytokines in Primary Cell Culture Secretome Using Targeted Mass Spectrometry.

Cytokines are commonly measured by immunoassays; however, these have limited multiplexing capacity, are costly, and can exhibit cross-reactivity. Multiple reaction monitoring (MRM) mass spectrometry is a robust method to quantify analytes with high specificity and multiplexing ability, hence we aimed to investigate its suitability as an alternative cost-effective method for cytokine measurement. Human keratinocyte conditioned media spiked with recombinant cytokines was used as an experimental system to evaluate sensitivity, linearity, and reproducibility of an MRM assay targeting 79 peptides representing 23 human cytokines. Our MRM method was able to identify 21 cytokines by two or more unique peptides and two cytokines by a single unique peptide. In a serum-free matrix, the median LOD and LOQ for cytokine peptides was 130 and 433 pg/mL, respectively. The presence of serum increased median LOD and LOQ by about 2.3-fold. The assay shows excellent replicate consistency with 8% intra- and 12% interday coefficient of variations. We found high pH reversed-phase fractionation a useful tool to increase assay sensitivity with the drawback of increasing its variability by approximately 10%. Overall, our results suggest utility of a multiplex cytokine MRM for routine measurement of secreted cytokines in cellular experiments under low serum conditions. Additional enrichment steps will be required in high complexity matrices such as serum.

Histone H3K4 monomethylation catalyzed by Trr and mammalian COMPASS-like proteins at enhancers is dispensable for development and viability.

Histone H3 lysine 4 monomethylation (H3K4me1) is an evolutionarily conserved feature of enhancer chromatin catalyzed by the COMPASS-like methyltransferase family, which includes Trr in Drosophila melanogaster and MLL3 (encoded by KMT2C) and MLL4 (encoded by KMT2D) in mammals. Here we demonstrate that Drosophila embryos expressing catalytically deficient Trr eclose and develop to productive adulthood. Parallel experiments with a trr allele that augments enzyme product specificity show that conversion of H3K4me1 at enhancers to H3K4me2 and H3K4me3 is also compatible with life and results in minimal changes in gene expression. Similarly, loss of the catalytic SET domains of MLL3 and MLL4 in mouse embryonic stem cells (mESCs) does not disrupt self-renewal. Drosophila embryos with trr alleles encoding catalytic mutants manifest subtle developmental abnormalities when subjected to temperature stress or altered cohesin levels. Collectively, our findings suggest that animal development can occur in the context of Trr or mammalian COMPASS-like proteins deficient in H3K4 monomethylation activity and point to a possible role for H3K4me1 on cis-regulatory elements in specific settings to fine-tune transcriptional regulation in response to environmental stress.