Propionibacterium (Cutibacterium) granulosum Extracellular DNase BmdE Targeting Propionibacterium (Cutibacterium) acnes Biofilm Matrix, a Novel Inter-Species Competition Mechanism

Acne vulgaris is the most common dermatological disorder worldwide affecting more than 80% of adolescents and young adults with a global prevalence of 231 million cases in 2019. The involvement of the skin microbiome disbalance in the pathophysiology of acne is recognized, especially regarding the relative abundance and diversity of Propionibacterium acnes a well-known dominant human skin commensal. Biofilms, where bacteria are embedded into a protective polymeric extracellular matrix, are the most prevalent life style for microorganisms. P. acnes and its biofilm-forming ability is believed to be a contributing factor in the development of acne vulgaris, the persistence of the opportunistic pathogen and antibiotic therapy failures. Degradation of the extracellular matrix is one of the strategies used by bacteria to disperse the biofilm of competitors. In this study, we report the identification of an endogenous extracellular nuclease, BmdE, secreted by Propionibacterium granulosum able to degrade P. acnes biofilm both in vivo and in vitro. This, to our knowledge, may represent a novel competitive mechanism between two closely related species in the skin. Antibiotics targeting P. acnes have been the mainstay in acne treatment. Extensive and long-term use of antibiotics has led to the selection and spread of resistant bacteria. The extracellular DNase BmdE may represent a new bio-therapeutical strategy to combat P. acnes biofilm in acne vulgaris.

Functional Analysis of Two Novel Streptococcus iniae Virulence Factors Using a Zebrafish Infection Model

Streptococcus iniae is a major fish pathogen that contributes to large annual losses in the aquaculture industry, exceeding US$100 million. It is also reported to cause opportunistic infections in humans. We have recently identified two novel S. iniae virulence factors, an extracellular nuclease (SpnAi) and a secreted nucleotidase (S5nAi), and verified their predicted enzymatic activities using recombinant proteins. Here, we report the generation of green fluorescent S. iniae spnAi and s5nAi deletion mutants and their evaluation in a transgenic zebrafish infection model. Our results show nuclease and nucleotidase activities in S. iniae could be attributed to SpnAi and S5nAi, respectively. Consistent with this, larvae infected with the deletion mutants demonstrated enhanced survival and bacterial clearance, compared to those infected with wild-type (WT) S. iniae. Deletion of spnAi and s5nAi resulted in sustained recruitment of neutrophils and macrophages, respectively, to the site of infection. We also show that recombinant SpnAi is able to degrade neutrophil extracellular traps (NETs) isolated from zebrafish kidney tissue. Our results suggest that both enzymes play an important role in S. iniae immune evasion and might present potential targets for the development of therapeutic agents or vaccines.

Recombineering inVibrio natriegens

Here, we show that λ-Red homologs found in theVibrio-associated SXT mobile element potentiate allelic exchange inV. natriegensby ~10,000-fold. Specifically, we show SXT-Beta (s065), SXT-Exo (s066), and λ-Gam proteins are sufficient to enable recombination of single- and double-stranded DNA with episomal and genomic loci. We characterize and optimize episomal oligonucleotide-mediated recombineering and demonstrate recombineering at genomic loci. We further show targeted genomic deletion of the extracellular nuclease genednsusing a double-stranded DNA cassette. Continued development of this recombination technology will advance high-throughput and large-scale genetic engineering efforts to domesticateV. natriegensand to investigate its rapid growth rate.