Protective effect of microbisporicin (NAI-107) against vancomycin resistant Enterococcus faecium infection in a Galleria mellonella model.

Increasing antimicrobial resistance in Enterococcus faecium necessitates the search for novel treatment agents, such as bacteriocins. In this study, we conducted an in vivo assessment of five bacteriocins, namely Lacticin Z, Lacticin Q, Garvicin KS (ABC), Aureocin A53 and Microbisporicin (NAI-107), against vanB-resistant Enterococcus faecium using a Galleria mellonella model. Our in vitro experiments demonstrated the efficacy of all five bacteriocins against vanB-resistant E. faecium with only NAI-107 demonstrating in vivo efficacy. Notably, NAI-107 exhibited efficacy across a range of tested doses, with the highest efficacy observed at a concentration of 16 µg/mL. Mortality rates in the group treated with 16 µg/mL NAI-107 were lower than those observed in the linezolid-treated group. These findings strongly suggest that NAI-107 holds promise as a potential alternative therapeutic agent for treating infections caused by resistant E. faecium and warrants further investigation.

Enhancing the antibacterial function of probiotic Escherichia coli Nissle: when less is more.

Probiotic bacteria confer multiple health benefits, including preventing the growth, colonization, or carriage of harmful bacteria in the gut. Bacteriocins are antibacterial peptides produced by diverse bacteria, and their production is tightly regulated and coordinated at the transcriptional level. A popular strategy for enhancing the antibacterial properties of probiotic bacteria is to retrofit them with the ability to overproduce heterologous bacteriocins. This is often achieved from non-native constitutive promoters or in response to host or pathogen signal from synthetic promoters. How the dysregulated overproduction of heterologous bacteriocins affects the fitness and antibacterial efficacy of the retrofitted probiotic bacteria is often overlooked. We have conferred the prototypical probiotic Escherichia coli strain Nissle (EcN) the ability to produce microcin C (McC) from the wild-type promoter and two mutant promoters that allow, relative to the wild-type promoter, high and low amounts of McC production. This was done by introducing specific changes to the sequence of the wild-type promoter driving transcription of the McC operon while ensuring that the modified promoters respond to native regulation. By studying the transcriptomic responses and antibacterial efficacy of the retrofitted EcN bacteria in a Galleria mellonella infection model of enterohemorrhagic E. coli, we show that EcN bacteria that produce the lowest amount of McC display the highest antibacterial efficacy with little-to-none undesired collateral impact on their fitness. The results highlight considerations researchers may take into account when retrofitting probiotic bacteria with heterogenous gene products for therapeutic, prophylactic, or diagnostic applications. Bacteria that resist killing by antibiotics are a major risk to modern medicine. The use of beneficial "probiotic" bacteria to make antibiotic-like compounds at the site of infection in the body is emerging as a popular alternative to the use of conventional antibiotics. A potential drawback of engineering probiotic bacteria in this way is that producing antibiotic-like compounds could impart undesired side effects on the performance of such bacteria, thereby compromising their intended use. This study highlights considerations researchers may take into account when engineering probiotic bacteria for therapeutic, prophylactic, or diagnostic applications.

Microbisporicin (NAI-107) protects Galleria mellonella from infection with Neisseria gonorrhoeae.

IMPORTANCE: We screened 66 bacteriocins to see if they exhibited anti-gonococcal activity. We found 12 bacteriocins with anti-gonococcal effects, and 4 bacteriocins showed higher anti-gonococcal activity. Three bacteriocins, lacticin Z, lacticin Q, and Garvicin KS (ABC), showed in vitro anti-gonococcal activity but no in vivo inhibitory effects against the Neisseria gonorrhoeae (WHO-P) isolate. On the other hand, NAI-107 showed in vivo anti-gonococcal activity. The findings suggest that NAI-107 is a promising alternative to treat gonorrhea infections.

Selective Bacteriocins: A Promising Treatment for Staphylococcus aureus Skin Infections Reveals Insights into Resistant Mutants, Vancomycin Resistance, and Cell Wall Alterations.

The emergence of antibiotic-resistant S. aureus has become a major public health concern, necessitating the discovery of new antimicrobial compounds. Given that the skin microbiome plays a critical role in the host defence against pathogens, the development of therapies that target the interactions between commensal bacteria and pathogens in the skin microbiome offers a promising approach. Here, we report the discovery of two bacteriocins, cerein 7B and cerein B4080, that selectively inhibit S. aureus without affecting S. epidermidis, a commensal bacterium on the skin. Our study revealed that exposure of S. aureus to these bacteriocins resulted in mutations in the walK/R two-component system, leading to a thickening of the cell wall visible by transmission electron microscopy and subsequent decreased sensitivity to vancomycin. Our findings prompt a nuanced discussion of the potential of those bacteriocins for selective targeting of S. aureus on the skin, given the emergence of resistance and co-resistance with vancomycin. The idea put forward implies that by preserving commensal bacteria, selective compounds could limit the emergence of resistance in pathogenic cells by promoting competition with remaining commensal bacteria, ultimately reducing chronical infections and limiting the spread of antibiotic resistance.

Deletion of QDR genes in a bioethanol-producing yeast strain reduces propagation of contaminating lactic acid bacteria.

Bacterial contaminations in yeast fermentation tanks are a recurring problem for the bioethanol production industry. Lactic acid bacteria (LAB), particularly of the genus Lactobacillus, are the most common contaminants. Their proliferation can reduce fermentation efficiency or even impose premature shutdown for cleaning. We have previously reported that laboratory yeast strains naturally excrete amino acids via transporters of the Drug: H+ Antiporter-1 (DHA1) family. This excretion allows yeast to cross-feed LAB, which are most often unable to grow without an external amino acid supply. Whether industrial yeast strains used in bioethanol production likewise promote LAB proliferation through cross-feeding has not been investigated. In this study, we first show that the yeast strain Ethanol Red used in ethanol production supports growth of Lactobacillus fermentum in an amino-acid-free synthetic medium. This effect was markedly reduced upon homozygous deletion of the QDR3 gene encoding a DHA1-family amino acid exporter. We further show that cultivation of Ethanol Red in a nonsterile sugarcane-molasses-based medium is associated with an increase in lactic acid due to LAB growth. When Ethanol Red lacked the QDR1, QDR2, and QDR3 genes, this lactic acid production was not observed and ethanol production was not significantly reduced. Our results indicate that Ethanol Red cultivated in synthetic or molasses medium sustains LAB proliferation in a manner that depends on its ability to excrete amino acids via Qdr transporters. They further suggest that using mutant industrial yeast derivatives lacking DHA1-family amino acid exporters may be a way to reduce the risk of bacterial contaminations during fermentation.

Promising Antimicrobial Activity and Synergy of Bacteriocins Against Mycobacterium tuberculosis.

In this study, we assessed the potential of bacteriocins and their in vitro synergistic effects in combination with anti-tuberculosis drugs against Mycobacterium tuberculosis. We evaluated the in vitro activity of chemically synthesized bacteriocins in combination with rifampicin (RIF), ofloxacin, and moxifloxacin against the reference M. tuberculosis H37Rv and a clinical-resistant strain. We first screened the bacteriocin PARAGEN collection and found active bacteriocins. We then determined their minimal inhibitory concentration (MIC), minimal bactericidal concentration, and their fractional inhibitory index by the checkerboard microdilution assay. Remarkably, we identified four bacteriocins with interesting antimycobacterial activity alone and in combinations with RIF, ofloxacin, and moxifloxacin, with significant reduction of the MIC that showed impressive synergistic effects against the susceptible and resistant clinical strains. In conclusion, our preliminary results show promising bacteriocins candidate used in a synergistic combination with anti-tuberculosis drugs and emphasize the need for combined therapy as a new strategy to enhance the activity of existing drugs, which may confer very promising therapeutic benefits against M. tuberculosis.

In vitro and in vivo production and split-intein mediated ligation (SIML) of circular bacteriocins.

Circular bacteriocins are antimicrobial peptides produced by bacteria that after synthesis undergo a head-to-tail circularization. Compared to their linear counterparts, circular bacteriocins are, in general, very stable to temperature and pH changes and more resistant to proteolytic enzymes, being considered as one of the most promising groups of antimicrobial peptides for their potential biotechnological applications. Up to now, only a reduced number of circular bacteriocins have been identified and fully characterized, although many operons potentially coding for new circular bacteriocins have been recently found in the genomes of different bacterial species. The production of these peptides is very complex and depends on the expression of different genes involved in their synthesis, circularization, and secretion. This complexity has greatly limited the identification and characterization of these bacteriocins, as well as their production in heterologous microbial hosts. In this work, we have evaluated a synthetic biology approach for the in vitro and in vivo production combined with a split-intein mediated ligation (SIML) of the circular bacteriocin garvicin ML (GarML). The expression of one single gene is enough to produce a protein that after intein splicing, circularizes in an active peptide with the exact molecular mass and amino acid sequence as native GarML. In vitro production coupled with SIML has been validated with other, well described and not yet characterized, circular bacteriocins. The results obtained suggest that this synthetic biology tool holds great potential for production, engineering, improving and testing the antimicrobial activity of circular bacteriocins.

In the Age of Synthetic Biology, Will Antimicrobial Peptides be the Next Generation of Antibiotics?

Antibiotics have changed human health and revolutionised medical practice since the Second World War. Today, the use of antibiotics is increasingly limited by the rise of antimicrobial-resistant strains. Additionally, broad-spectrum antibiotic activity is not adapted to maintaining a balanced microbiome essential for human health. Targeted antimicrobials could overcome these two drawbacks. Although the rational design of targeted antimicrobial molecules presents a formidable challenge, in nature, targeted genetically encoded killing molecules are used by microbes in their natural ecosystems. The use of a synthetic biology approach allows the harnessing of these natural functions. In this commentary article we illustrate the potential of applying synthetic biology towards bacteriocins to design a new generation of antimicrobials.

PARAGEN 1.0: A Standardized Synthetic Gene Library for Fast Cell-Free Bacteriocin Synthesis.

The continuous emergence of microbial resistance to our antibiotic arsenal is widely becoming recognized as an imminent threat to global human health. Bacteriocins are antimicrobial peptides currently under consideration as real alternatives or complements to common antibiotics. These peptides have been much studied, novel bacteriocins are regularly reported and several genomic databases on these peptides are currently updated. Despite this, to our knowledge, a physical collection of bacteriocins that would allow testing and comparing them for different applications does not exist. Rapid advances in synthetic biology in combination with cell-free protein synthesis technologies offer great potential for fast protein production. Based on the amino acid sequences of the mature peptide available in different databases, we have built a bacteriocin gene library, called PARAGEN 1.0, containing all the genetic elements required for in vitro cell-free peptide synthesis. Using PARAGEN 1.0 and a commercial kit for cell-free protein synthesis we have produced 164 different bacteriocins. Of the bacteriocins synthesized, 54% have shown antimicrobial activity against at least one of the indicator strains tested, including Gram-positive and Gram-negative bacteria representing commonly used lab strains, industrially relevant microorganisms, and known pathogens. This bacteriocin collection represents a streamlined pipeline for selection, production, and screening of bacteriocins as well as a reservoir of ready-to-use antimicrobials against virtually any class of relevant bacteria.

Mobilization of Microbiota Commensals and Their Bacteriocins for Therapeutics.

With the specter of resurgence of pathogens due to the propagation of antibiotic-resistance genes, innovative antimicrobial strategies are needed. In this review, we summarize the beneficial aspects of bacteriocins, a set of miscellaneous peptide-based bacterium killers, compared with classical antibiotics, and emphasize their use in cocktails to curb the emergence of new resistance. We highlight that their prey spectrum, their molecular malleability, and their multiple modes of production might lead to specific and personalized treatments to prevent systemic disorders. Complementarily, we discuss how we might exploit prevailing bacterial commensals, such as Streptococcus salivarius, and deliberately mobilize their bacteriocin arsenal 'on site' to cure multiresistant infections or finely reshape the endogenous microbiota for prophylaxis purposes.

Alpha-fetoprotein controls female fertility and prenatal development of the gonadotropin-releasing hormone pathway through an antiestrogenic action.

It has been shown previously that female mice homozygous for an alpha-fetoprotein (AFP) null allele are sterile as a result of anovulation, probably due to a defect in the hypothalamic-pituitary axis. Here we show that these female mice exhibit specific anomalies in the expression of numerous genes in the pituitary, including genes involved in the gonadotropin-releasing hormone pathway, which are underexpressed. In the hypothalamus, the gonadotropin-releasing hormone gene, Gnrh1, was also found to be down-regulated. However, pituitary gene expression could be normalized and fertility could be rescued by blocking prenatal estrogen synthesis using an aromatase inhibitor. These results show that AFP protects the developing female brain from the adverse effects of prenatal estrogen exposure and clarify a long-running debate on the role of this fetal protein in brain sexual differentiation.

Alpha-fetoprotein protects the developing female mouse brain from masculinization and defeminization by estrogens.

Two clearly opposing views exist on the function of alpha-fetoprotein (AFP), a fetal plasma protein that binds estrogens with high affinity, in the sexual differentiation of the rodent brain. AFP has been proposed to either prevent the entry of estrogens or to actively transport estrogens into the developing female brain. The availability of Afp mutant mice (Afp(-/-)) now finally allows us to resolve this longstanding controversy concerning the role of AFP in brain sexual differentiation, and thus to determine whether prenatal estrogens contribute to the development of the female brain. Here we show that the brain and behavior of female Afp(-/-) mice were masculinized and defeminized. However, when estrogen production was blocked by embryonic treatment with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione, the feminine phenotype of these mice was rescued. These results clearly demonstrate that prenatal estrogens masculinize and defeminize the brain and that AFP protects the female brain from these effects of estrogens.

Alpha-fetoprotein, the major fetal serum protein, is not essential for embryonic development but is required for female fertility.

The alpha-fetoprotein gene (Afp) is a member of a multigenic family that comprises the related genes encoding albumin, alpha-albumin, and vitamin D binding protein. The biological role of this major embryonic serum protein is unknown although numerous speculations have been made. We have used gene targeting to show that AFP is not required for embryonic development. AFP null embryos develop normally, and individually transplanted homozygous embryos can develop in an AFP-deficient microenvironment. Whereas mutant homozygous adult males are viable and fertile, AFP null females are infertile. Our analyses of these mice indicate that the defect is caused by a dysfunction of the hypothalamic/pituitary system, leading to anovulation.

Stimulation of the alpha-fetoprotein promoter by unliganded thyroid hormone receptor in association with protein deacetylation.

alpha-Fetoprotein (AFP) is a serum protein expressed during fetal life, the expression of which is shut off after birth. The activity of the mouse Afp gene promoter region comprised between -80 and -38 bp is regulated by the thyroid hormone receptor (T3R): negatively in the presence of T3 and positively in the absence of T3. The stimulating effect of unliganded T3R is, unexpectedly, antagonized by cofactors that have histone-acetyl-transferase activity, or by sodium butyrate, which inhibits histone acetylases (HDACs). The unliganded T3R stimulating activity effect is thus associated with protein deacetylation, contrary to the usual situation. In combination with previous results, our observations suggest that T3-mediated down regulation of the Afp promoter is due to T3-induced protein acetylation leading to loss of a nucleosomal structure (required for promoter activity) and chromatin opening.