Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes

Effects of the Staphylococcus aureus and Staphylococcus epidermidis Secretomes Isolated from the Skin Microbiota of Atopic Children on CD4+ T Cell Activation.

Interactions between the immune system and skin bacteria are of major importance in the pathophysiology of atopic dermatitis (AD), yet our understanding of them is limited. From a cohort of very young AD children (1 to 3 years old), sensitized to Dermatophagoides pteronyssinus allergens (Der p), we conducted culturomic analysis of skin microbiota, cutaneous transcript profiling and quantification of anti-Der p CD4+ T cells. This showed that the presence of S. aureus in inflamed skin of AD patients was associated with a high IgE response, increased expression of inflammatory and Th2/Th22 transcripts and the prevalence of a peripheral Th2 anti-Der p response. Monocyte-derived dendritic cells (moDC) exposed to the S. aureus and S. epidermidis secretomes were found to release pro-inflammatory IFN-γ and anti-inflammatory IL-10, respectively. Allogeneic moDC exposed to the S. aureus secretome also induced the proliferation of CD4+ T cells and this effect was counteracted by concurrent exposure to the S. epidermidis secretome. In addition, whereas the S. epidermidis secretome promoted the activity of regulatory T cells (Treg) in suppressing the proliferation of conventional CD4+ T cells, the Treg lost this ability in the presence of the S. aureus secretome. We therefore conclude that S. aureus may cause and promote inflammation in the skin of AD children through concomitant Th2 activation and the silencing of resident Treg cells. Commensals such as S. epidermidis may counteract these effects by inducing the release of IL-10 by skin dendritic cells

Transformation of acinetobacter baumannii: Electroporation

Although the pan and the core genome of Acinetobacter baumannii and its essential genes are relatively well characterized, functional characterization of these genes has not paralleled the genome-level studies. However, recently developed genetic tools and optimized protocols are poised to accelerate genetic manipulation of A. baumannii. Transferring exogenous DNA into the cytosol of bacteria cells is a critical step in genetic characterizations. Conjugation is restricted to the transfer of DNA from one bacterial cell to another, and only a portion of A. baumannii clinical isolates are naturally competent. Electroporation, which is thought to transiently create aqueous pores in the membrane, is a preferred method in transferring exogenous DNA as it does not have such limitations. Several factors contribute to efficiency of electroporation and often need to be empirically optimized to maximize efficiency of this procedure. Here we provide an optimized electroporation protocol and guidance for electroporation of clinical MDR isolates of A. baumannii

IL-4-producing peripheral T CD4⁺ cells against Der p allergens are increased in AD children compared to IFN-γ- producing T CD4⁺ cells.

<p>(A) IFN-γ and IL-4 ELISPot assays (spot forming units/10⁶ T cells) were performed on peripheral blood from non-AD (N = 14) and AD (N = 15) children in response to crude extracts of Der p. (B) Ratio of IL-4 <i>vs</i> IFN-γ CD4⁺ T cell spots relative to titers of total or Der p1 specific IgE antibodies (kU/ml) in AD patients. Mann-Whitney, mean values with SEM are shown, p*<0.05, p**<0.01.</p