Manfred Biebl

Structure of the Receptor-Binding Protein of Bacteriophage Det7: a Podoviral Tail Spike in a Myovirus

ABSTRACT A new Salmonella enterica phage, Det7, was isolated from sewage and shown by electron microscopy to belong to the Myoviridae morphogroup of bacteriophages. Det7 contains a 75-kDa protein with 50% overall sequence identity to the tail spike endorhamnosidase of podovirus P22. Adsorption of myoviruses to their bacterial hosts is normally mediated by long and short tail fibers attached to a contractile tail, whereas podoviruses do not contain fibers but attach to host cells through stubby tail spikes attached to a very short, noncontractile tail. The amino-terminal 150 residues of the Det7 protein lack homology to the P22 tail spike and are probably responsible for binding to the base plate of the myoviral tail. Det7 tail spike lacking this putative particle-binding domain was purified from Escherichia coli, and well-diffracting crystals of the protein were obtained. The structure, determined by molecular replacement and refined at a 1.6-Å resolution, is very similar to that of bacteriophage P22 tail spike. Fluorescence titrations with an octasaccharide suggest Det7 tail spike to bind its receptor lipopolysaccharide somewhat less tightly than the P22 tail spike. The Det7 tail spike is even more resistant to thermal unfolding than the already exceptionally stable homologue from P22. Folding and assembly of both trimeric proteins are equally temperature sensitive and equally slow. Despite the close structural, biochemical, and sequence similarities between both proteins, the Det7 tail spike lacks both carboxy-terminal cysteines previously proposed to form a transient disulfide during P22 tail spike assembly. Our data suggest receptor-binding module exchange between podoviruses and myoviruses in the course of bacteriophage evolution.

Art-175 Is a Highly Efficient Antibacterial against Multidrug-Resistant Strains and Persisters of Pseudomonas aeruginosa

ABSTRACT Artilysins constitute a novel class of efficient enzyme-based antibacterials. Specifically, they covalently combine a bacteriophage-encoded endolysin, which degrades the peptidoglycan, with a targeting peptide that transports the endolysin through the outer membrane of Gram-negative bacteria. Art-085, as well as Art-175, its optimized homolog with increased thermostability, are each composed of the sheep myeloid 29-amino acid (SMAP-29) peptide fused to the KZ144 endolysin. In contrast to KZ144, Art-085 and Art-175 pass the outer membrane and kill Pseudomonas aeruginosa, including multidrug-resistant strains, in a rapid and efficient (∼5 log units) manner. Time-lapse microscopy confirms that Art-175 punctures the peptidoglycan layer within 1 min, inducing a bulging membrane and complete lysis. Art-175 is highly refractory to resistance development by naturally occurring mutations. In addition, the resistance mechanisms against 21 therapeutically used antibiotics do not show cross-resistance to Art-175. Since Art-175 does not require an active metabolism for its activity, it has a superior bactericidal effect against P. aeruginosa persisters (up to >4 log units compared to that of the untreated controls). In summary, Art-175 is a novel antibacterial that is well suited for a broad range of applications in hygiene and veterinary and human medicine, with a unique potential to target persister-driven chronic infections.

‘Artilysation’ of endolysin λSa2lys strongly improves its enzymatic and antibacterial activity against streptococci

Endolysins constitute a promising class of antibacterials against Gram-positive bacteria. Recently, endolysins have been engineered with selected peptides to obtain a new generation of lytic proteins, Artilysins, with specific activity against Gram-negative bacteria. Here, we demonstrate that artilysation can also be used to enhance the antibacterial activity of endolysins against Gram-positive bacteria and to reduce the dependence on external conditions. Art-240, a chimeric protein of the anti-streptococcal endolysin λSa2lys and the polycationic peptide PCNP, shows a similar species specificity as the parental endolysin, but the bactericidal activity against streptococci increases and is less affected by elevated NaCl concentrations and pH variations. Time-kill experiments and time-lapse microscopy demonstrate that the killing rate of Art-240 is approximately two-fold higher compared to wildtype endolysin λSa2lys, with a reduction in viable bacteria of 3 log units after 10 min. In addition, lower doses of Art-240 are required to achieve the same bactericidal effect.

Ambivalent bacteriophages of different species active on Escherichia coli K12 and Salmonella sps. strains

A study was made of several bacteriophages (including phages U2 and LB related to T-even phages of Escherichia coli) that grow both on E. coli K12 and on some Salmonella strains. Such phages were termed ambivalent. T-even ambivalent phages (U2 and LB) are rare and have a limited number of hosts among Salmonella strains. U2 and LB are similar to canonical E. coli-specific T-even phages in morphological type and size of the phage particle and in reaction with specific anti-T4 serum. Phages U2 and LB have identical sets of structural proteins, some of which are similar in size to structural proteins of phages T2 and T4. DNA restriction patterns of phages U2 and LB differ from each other and from those of T2 and T4. Still, DNAs of all four phages have considerable homology. Unexpectedly, phages U2 and LB grown on Salmonella bongori were unstable during centrifugation in a CsCl gradient. Ambivalent bacteriophages were found in species other than T-even phages and were similar in morphotype to lambdoid and other E. coli phages. One of the ambivalent phages was highly similar to well-known Felix01, which is specific for Salmonella. Ambivalent phages can be used to develop a new set for phage typing in Salmonella. An obvious advantage is that ambivalent phages can be reproduced in the E. coli K12 laboratory strain, which does not produce active temperature phages. Consequently, the resulting typing phage preparation is devoid of an admixture of temperate phages, which are common in Salmonella. The presence of temperate phages in phage-typing preparations may cause false-positive results in identifying specific Salmonella strains isolated from the environment or salmonellosis patients. Ambivalent phages are potentially useful for phage therapy and prevention of salmonellosis in humans and animals.

Sample Preparation – An Essential Prerequisite for High-Quality Bacteria Detection

Rapid microbial testing is more and more preferred worldwide. Conventional time-consuming methods with detection times taking up to several days are being replaced by rapid tests that take only a few hours. With the development of new, rapid, and accurate methods for the detection of bacterial contaminants, the requirements for sample preparation techniques are more and more challenging. In fact, sample preparation is the critical step with respect to the applicability of novel methods. Sample preparation comprises sampling/sample drawing, sample handling, and sample preparation. To fulfil the demands of modern microbiology the ideal procedure should permit rapidly providing the processed sample in a small volume which contains the analyte in the highest concentration possible. The analyte has to be free of substances interfering with the detection method to be finally applied. Additionally, sample processing procedures used should not result in any loss of the bacterial analyte, thereby enabling quantitative measurements.

Inhibitory and bactericidal effect of Artilysin ® Art-175 against colistin-resistant mcr-1 -positive Escherichia coli isolates

The number of mcr-1-positive isolates has increased tremendously since the first report of the plasmid-mediated colistin– resistance mechanism [1]. Isolates with the transferable gene have been identified in (food) animals, various types of meat, vegetables and humans in more than 30 countries [2]. Consequently, the Chinese government has forbidden the use of colistin as a growth promotor for the veterinary sector. The European Medicines Agency now suggests to classify colistin in a higher-risk category and to restrict the use in animals to a maximum 5 mg/ population correction unit [2]. However, in humans, colistin is predominantly used as a last resort parenteral drug against highly resistant bacteria, such as carbapenem-resistant Enterobacteriaceae and Acinetobacter spp. A total ban on colistin in human health is thus not conceivable, but there is an increasing risk that pan– drug-resistant bacteria will emerge. As such, the development of new agents effective against resistant Gram-negative pathogens is important. Artilysin®s are new, engineered, enzyme-based experimental therapeutics active against Gram-negative and Gram-positive pathogens [3]. Specifically, Artilysin® Art-175 consists of an outer membrane (OM) destabilizing peptide fused to an endolysin, which is a peptidoglycan hydrolase encoded by a bacterial virus. Upon application, the OM is first locally destabilized by the peptide, after which Art175 is transferred through the OM and the endolysin moiety extensively degrades the peptidoglycan layer, resulting in membrane bulging and osmotic cell lysis [4]. Based on high clinical impact and technical feasibility, endolysin-based antibacterials, such as Artilysin®s, have recently been classified as antibiotic alternatives with the highest potential as therapeutics [5]. The bactericidal effect of Artilysin® Art-175 against Gramnegative pathogens has already been demonstrated for resistant and persistent cells of Pseudomonas aeruginosa and Acinetobacter baumannii. Moreover, neither resistance development through genetic alterations, nor cross-resistance with existing antibiotic resistance mechanisms has been observed [3,4]. However, both colistin and Art-175 target anionic lipopolysaccharide (LPS) molecules, which leads to a local disturbance in the OM of Gramnegative bacteria. Furthermore, colistin resistance is caused by phosphoethanolamine substitutions on the phosphate groups of the lipid A moiety of the LPS molecules, caused by the mcr-1– encoded phosphoethanolaminotransferase. Hence, cross-resistance could potentially occur. The objectives of this study, therefore, were (i) to evaluate the potential of Art-175 against isolates that have high risk of developing pan-drug resistance; and (ii) to exclude a potential cross–resistance between colistin and Art-175. During a period of three months in 2016, faeces and organ samples from diarrheic swine and cattle from Bavarian farms were monitored for the presence of Escherichia coli. The species of the obtained isolates (n = 19) were confirmed by MALDI-TOF MS fingerprinting (Microflex LT, Bruker Daltonik, Bremen, Germany) using the MALDI Biotyper software, version 4.0.0.1. Confirmed isolates were tested for colistin susceptibility by broth microdilution (Sifin Diagnostics, Berlin, Germany) according to the guidelines of the CLSI. Eleven isolates were found to be colistin-resistant (Table 1). Plasmid DNA was isolated from all colistin-resistant isolates and analysed by polymerase chain reaction (PCR) for the presence of the mcr-1