Novel BRICHOS-Related Antimicrobial Peptides from the Marine Worm Heteromastus filiformis: Transcriptome Mining, Synthesis, Biological Activities, and Therapeutic Potential.

Marine polychaetes represent an extremely rich and underexplored source of novel families of antimicrobial peptides (AMPs). The rapid development of next generation sequencing technologies and modern bioinformatics approaches allows us to apply them for characterization of AMP-derived genes and the identification of encoded immune-related peptides with the aid of genome and transcriptome mining. Here, we describe a universal bioinformatic approach based on the conserved BRICHOS domain as a search query for the identification of novel structurally unique AMP families in annelids. In this paper, we report the discovery of 13 novel BRICHOS-related peptides, ranging from 18 to 91 amino acid residues in length, in the cosmopolitan marine worm Heteromastus filiformis with the assistance of transcriptome mining. Two characteristic peptides with a low homology in relation to known AMPs-the α-helical amphiphilic linear peptide, consisting of 28 amino acid residues and designated as HfBRI-28, and the 25-mer ß-hairpin peptide, specified as HfBRI-25 and having a unique structure stabilized by two disulfide bonds-were obtained and analyzed as potential antimicrobials. Interestingly, both peptides showed the ability to kill bacteria via membrane damage, but mechanisms of their action and spectra of their activity differed significantly. Being non-cytotoxic towards mammalian cells and stable to proteolysis in the blood serum, HfBRI-25 was selected for further in vivo studies in a lethal murine model of the Escherichia coli infection, where the peptide contributed to the 100% survival rate in animals. A high activity against uropathogenic strains of E. coli (UPEC) as well as a strong ability to kill bacteria within biofilms allow us to consider the novel peptide HfBRI-25 as a promising candidate for the clinical therapy of urinary tract infections (UTI) associated with UPEC.

Design of Protegrin-1 Analogs with Improved Antibacterial Selectivity.

Protegrin-1 (PG-1) is a cationic ß-hairpin pore-forming antimicrobial peptide having a membranolytic mechanism of action. It possesses in vitro a potent antimicrobial activity against a panel of clinically relevant MDR ESKAPE pathogens. However, its extremely high hemolytic activity and cytotoxicity toward mammalian cells prevent the further development of the protegrin-based antibiotic for systemic administration. In this study, we rationally modulated the PG-1 charge and hydrophobicity by substituting selected residues in the central ß-sheet region of PG-1 to design its analogs, which retain a high antimicrobial activity but have a reduced toxicity toward mammalian cells. In this work, eight PG-1 analogs with single amino acid substitutions and five analogs with double substitutions were obtained. These analogs were produced as thioredoxin fusions in Escherichia coli. It was shown that a significant reduction in hemolytic activity without any loss of antimicrobial activity could be achieved by a single amino acid substitution, V16R in the C-terminal ß-strand, which is responsible for the PG-1 oligomerization. As the result, a selective analog with a ≥30-fold improved therapeutic index was obtained. FTIR spectroscopy analysis of analog, [V16R], revealed that the peptide is unable to form oligomeric structures in a membrane-mimicking environment, in contrast to wild-type PG-1. Analog [V16R] showed a reasonable efficacy in septicemia infection mice model as a systemic antibiotic and could be considered as a promising lead for further drug design.

Hetero-Pathogenic O181:H4 EAHEC Strain of Sequence Type ST678 Associated with Hemolytic-Uremic Syndrome in Schoolchildren in Russia.

BACKGROUND: In the last decade, the importance of hetero-pathogenic enteroaggregative Shiga-toxin-producing E. coli for public health has increased. Recently, we described the genetic background of the EAHEC O181:H4 strain of ST678 carrying the stx2 gene in prophage and five plasmids, including the plasmid-carrying aggR and aaiC genes. Here, we present the morphological and enzymatic characteristics of this strain, as well as susceptibility to antimicrobials, biofilm formation, etc. Methods: Bacterial morphology was studied using an electron microscope. Susceptibility to antimicrobials was determined using the microdilution method. Cytotoxicity was estimated in Vero cells. Virulence was studied on mice. RESULTS: The morphological and enzymatic properties of the hetero-pathogenic EAHEC strain were typical for E. coli; electron microscopy revealed the specific flagella. The strain was susceptible to most antibiotics and disinfectants but resistant to ampicillin and ciprofloxacin and showed a high degree of biofilm formation. Cytotoxicity towards Vero cells was estimated as 80%. CONCLUSIONS: The emergence of a new O181:H4 EAHEC strain poses a potential threat to humans because of the virulence potential that must be taken into account in the epidemiological analysis of outbreaks and sporadic cases of foodborne infections associated with hemolytic-uremic syndrome.

Novel ß-Hairpin Peptide from Marine Polychaeta with a High Efficacy against Gram-Negative Pathogens.

In recent years, new antibiotics targeting multidrug resistant Gram-negative bacteria have become urgently needed. Therefore, antimicrobial peptides are considered to be a novel perspective class of antibacterial agents. In this study, a panel of novel BRICHOS-related ß-hairpin antimicrobial peptides were identified in transcriptomes of marine polychaeta species. Two of them-abarenicin from Abarenicola pacifica and UuBRI-21 from Urechis unicinctus-possess strong antibacterial potential in vitro against a wide panel of Gram-negative bacteria including drug-resistant strains. Mechanism of action assays demonstrate that peptides disrupt bacterial and mammalian membrane integrity. Considering the stronger antibacterial potential and a low ability of abarenicin to be bound by components of serum, this peptide was selected for further modification. We conducted an alanine and arginine scanning of abarenicin by replacing individual amino acids and modulating hydrophobicity so as to improve its antibacterial potency and membrane selectivity. This design approach allowed us to obtain the Ap9 analog displaying a high efficacy in vivo in the mice septicemia and neutropenic mice peritonitis models. We demonstrated that abarenicin analogs did not significantly induce bacterial resistance after a four-week selection experiment and acted on different steps of the biofilm formation: (a) killing bacteria at their planktonic stage and preventing biofilm formation and (b) degrading pre-formed biofilm and killing embedded bacteria. The potent antibacterial and antibiofilm activity of the abarenicin analog Ap9 with its high efficacy in vivo against Gram-negative infection in mice models makes this peptide an attractive candidate for further preclinical investigation.

Comparative analysis of Klebsiella pneumoniae strains isolated in 2012-2016 that differ by antibiotic resistance genes and virulence genes profiles.

The antibacterial resistance and virulence genotypes and phenotypes of 148 non-duplicate Klebsiella pneumoniae strains collected from 112 patients in Moscow hospitals in 2012-2016 including isolates from the respiratory system (57%), urine (30%), wounds (5%), cerebrospinal fluid (4%), blood (3%), and rectal swab (1%) were determined. The majority (98%) were multidrug resistant (MDR) strains carrying blaSHV (91%), blaCTX-M (74%), blaTEM (51%), blaOXA (38%), and blaNDM (1%) beta-lactamase genes, class 1 integrons (38%), and the porin protein gene ompK36 (96%). The beta-lactamase genes blaTEM-1, blaSHV-1, blaSHV-11, blaSHV-110, blaSHV-190, blaCTX-M-15, blaCTX-M-3, blaCTX-M-55, blaOXA-48, blaOXA-244, and blaNDM-1 were detected; class 1 integron gene cassette arrays (aadA1), (dfrA7), (dfrA1-orfC), (aadB-aadA1), (dfrA17-aadA5), and (dfrA12-orfF-aadA2) were identified. Twenty-two (15%) of clinical K. pneumoniae strains had hypermucoviscous (HV) phenotype defined as string test positive. The rmpA gene associated with HV phenotype was detected in 24% of strains. The intrapersonal mutation of rmpA gene (deletion of one nucleotide at the polyG tract) was a reason for negative hypermucoviscosity phenotype and low virulence of rmpA-positive K. pneumoniae strain KPB584. Eighteen virulent for mice strains with LD50 ≤ 104 CFU were attributed to sequence types ST23, ST86, ST218, ST65, ST2174, and ST2280 and to capsular types K1, K2, and K57. This study is the first report about hypervirulent K. pneumoniae strain KPB2580-14 of ST23K1 harboring extended-spectrum beta-lactamase CTX-M-15 and carbapenemase OXA-48 genes located on pCTX-M-15-like and pOXA-48-like plasmids correspondingly.

Draft Genome Sequences of Eight Staphylococcus aureus Strains Isolated during Foodborne Outbreaks.

We report here the draft genome sequences of eight Staphylococcus aureus strains isolated during three large food poisoning outbreaks in the Russian Federation. The strains were collected from clinical specimens and various foodstuff samples.

Draft Genome Sequences of Exfoliative Toxin A-Producing Staphylococcus aureus Strains B-7772 and B-7777 (CC8/ST2993) and B-7774 (CC15/ST2126), Isolated in a Maternity Hospital in the Central Federal District of Russia.

Staphylococcus aureus clonal complex 8 (CC8) has not been associated with staphylococcal scalded-skin syndrome (SSSS) in newborns and exfoliative toxin genes. Here, we report the draft genome sequences of exfoliative toxin A-producing B-7772, B-7777 (both CC8), and B-7774 (CC15) strains associated with SSSS in newborns.

Lytic activity of the staphylolytic Twort phage endolysin CHAP domain is enhanced by the SH3b cell wall binding domain.

Increases in the prevalence of antibiotic-resistant strains of Staphylococcus aureus have elicited efforts to develop novel antimicrobials to treat these drug-resistant pathogens. One potential treatment repurposes the lytic enzymes produced by bacteriophages as antimicrobials. The phage Twort endolysin (PlyTW) harbors three domains, a cysteine, histidine-dependent amidohydrolases/peptidase domain (CHAP), an amidase-2 domain and a SH3b-5 cell wall binding domain (CBD). Our results indicate that the CHAP domain alone is necessary and sufficient for lysis of live S. aureus, while the amidase-2 domain is insufficient for cell lysis when provided alone. Loss of the CBD results in ∼10X reduction of enzymatic activity in both turbidity reduction and plate lysis assays compared to the full length protein. Deletion of the amidase-2 domain resulted in a protein (PlyTW Δ172-373) with lytic activity that exceeded the activity of the full length construct in both the turbidity reduction and plate lysis assays. Addition of Ca(2+) enhanced the turbidity reduction activity of both the full length protein and truncation constructs harboring the CHAP domain. Chelation by addition of EDTA or the addition of zinc inhibited the activity of all PlyTW constructs.

Staphylococcal phage 2638A endolysin is lytic for Staphylococcus aureus and harbors an inter-lytic-domain secondary translational start site.

Staphylococcus aureus is a notorious pathogen highly successful at developing resistance to virtually all antibiotics to which it is exposed. Staphylococcal phage 2638A endolysin is a peptidoglycan hydrolase that is lytic for S. aureus when exposed externally, making it a new candidate antimicrobial. It shares a common protein organization with more than 40 other reported staphylococcal peptidoglycan hydrolases. There is an N-terminal M23 peptidase domain, a mid-protein amidase 2 domain (N-acetylmuramoyl-L-alanine amidase), and a C-terminal SH3b cell wall-binding domain. It is the first phage endolysin reported with a secondary translational start site in the inter-lytic-domain region between the peptidase and amidase domains. Deletion analysis indicates that the amidase domain confers most of the lytic activity and requires the full SH3b domain for maximal activity. Although it is common for one domain to demonstrate a dominant activity over the other, the 2638A endolysin is the first in this class of proteins to have a high-activity amidase domain (dominant over the N-terminal peptidase domain). The high activity amidase domain is an important finding in the quest for high-activity staphylolytic domains targeting novel peptidoglycan bonds.

Staphylococcus haemolyticus prophage ΦSH2 endolysin relies on cysteine, histidine-dependent amidohydrolases/peptidases activity for lysis 'from without'.

Staphylococcus aureus is an important pathogen, with methicillin-resistant (MRSA) and multi-drug resistant strains becoming increasingly prevalent in both human and veterinary clinics. S. aureus causing bovine mastitis yields high annual losses to the dairy industry. Conventional treatment of mastitis by broad range antibiotics is often not successful and may contribute to development of antibiotic resistance. Bacteriophage endolysins present a promising new source of antimicrobials. The endolysin of prophage ΦSH2 of Staphylococcus haemolyticus strain JCSC1435 (ΦSH2 lysin) is a peptidoglycan hydrolase consisting of two catalytic domains (CHAP and amidase) and an SH3b cell wall binding domain. In this work, we demonstrated its lytic activity against live staphylococcal cells and investigated the contribution of each functional module to bacterial lysis by testing a series of deletion constructs in zymograms and turbidity reduction assays. The CHAP domain exhibited three-fold higher activity than the full length protein and optimum activity in physiological saline. This activity was further enhanced by the presence of bivalent calcium ions. The SH3b domain was shown to be required for full activity of the complete ΦSH2 lysin. The full length enzyme and the CHAP domain showed activity against multiple staphylococcal strains, including MRSA strains, mastitis isolates, and CoNS.