Novel Host Recognition Mechanism of the K1 Capsule-Specific Phage of Escherichia coli : Capsular Polysaccharide as the First Receptor and Lipopolysaccharide as the Secondary Receptor

K1 capsule-specific phages of Escherichia coli have been reported in recent years, but the molecular mechanism involved in host recognition of these phages remains unknown. In this study, the interactions between PNJ1809-36, a new K1-specific phage and its host bacteria E. coli DE058, were investigated. A transposon mutation library was used to screen for receptor-related genes. Gene deletion, lysis curve determination, plaque formation test, adsorption assay and inhibition assay of phage by lipopolysaccharide (LPS) showed that capsular polysaccharide (CPS) was the first receptor for the initial adsorption of PNJ1809-36 to E. coli DE058 and LPS was a secondary receptor for the irreversible binding of the phage. The penultimate galactose in the outer core was identified as the specific binding region on LPS. Through antibody blocking assay, fluorescence labeling and high-performance gel permeation chromatography (HPGPC), the tail protein ORF261 of phage PNJ1809-36 was identified as the receptor binding protein on CPS. Given these findings, we propose a model for the recognition process of phage PNJ1809-36 on E. coli DE058: The phage PNJ1809-36 tail protein ORF261 recognizes and adsorbs to the K1 capsule; then the K1 capsule is partially degraded, exposing the active site of LPS which is recognized by phage PNJ1809-36. This model provides insight into the molecular mechanisms between K1-specific phages and their host bacteria. IMPORTANCE It has been speculated that CPS is the main receptor of K1-specific phages belonging to Siphoviridae . In recent years, a new type of K1-specific phage belonging to Myoviridae has been reported, but its host recognition mechanisms remain unknown. Here, we studied the interactions between PNJ1809-36, a new type of K1 phage, and its host bacteria E. coli DE058. Our research showed that the phage initially adsorbed to the K1 capsule mediated by ORF261 and then bound to the penultimate galactose of LPS to begin the infection process.

Analysing Parallel Strategies to Alter the Host Specificity of Bacteriophage T7

The recognition and binding of host bacteria by bacteriophages is most often enabled by a highly specific receptor–ligand type of interaction, with the receptor-binding proteins (RBPs) of phages being the primary determinants of host specificity. Specifically modifying the RBPs could alter or extend the host range of phages otherwise exhibiting desired phenotypic properties. This study employed two different strategies to reprogram T7 phages ordinarily infecting commensal K12 Escherichia coli strains to infect pathogen-associated K1-capsule-expressing strains. The strategies were based on either plasmid-based homologous recombination or bacteriophage recombineering using electroporated DNA (BRED). Our work pursued the construction of two genetic designs: one replacing the gp17 gene of T7, the other replacing gp11, gp12, and gp17 of T7 with their K1F counterparts. Both strategies displayed successful integration of the K1F sequences into the T7 genome, detected by PCR screening. Multiple methods were utilised to select or enrich for chimeric phages incorporating the K1F gp17 alone, including trxA, host-specificity, and CRISPR-Cas-based selection. Irrespective of the selection method, the above strategy yielded poorly reproducible phage propagation on the new host, indicating that the chimeric phage was less fit than the wild type and could not promote continual autonomous reproduction. Chimeric phages obtained from BRED incorporating gp11-12 and gp17, however, all displayed infection in a 2-stage pattern, indicating the presence of both K1F and T7 phenotypes. This study shows that BRED can be used as a tool to quickly access the potential of new RBP constructs without the need to engineer sustainably replicating phages. Additionally, we show that solely repurposing the primary RBP is, in some cases, insufficient to produce a viable chimeric phage.

orf6 and orf10 in Prophage phiv142-3 Enhance the Iron-Acquisition Ability and Resistance of Avian Pathogenic Escherichia coli Strain DE142 to Serum

Avian pathogenic Escherichia coli (APEC), an extraintestinal pathogenic E. coli (ExPEC), is the causative agent of avian colibacillosis, a disease that causes huge economic losses in the poultry industry and is characterized by infection through respiratory tract colonization followed by bacteraemia. A previous study in our lab demonstrated that phiv142-3 enhanced the survival ability of APEC strain DE142 in chickens serum. However, the mechanism of this affect has not been completely revealed. Here, we analyzed the transcriptional level of the prophage phiv142-3 region in DE142 when grown in chicken serum. Several upregulated genes attracted our attention, and a series of mutants were constructed. Deletion of orf6 or orf10 from phiv142-3 led to lower yields compared with WT after cultivation in serum for 10 h (P < 0.05). Furthermore, avian infection assays showed that compared with WT, the bacterial loads in blood and heart tissue of chickens challenged with DE142Δorf6 were decreased to 3.9 and 13%, while the bacterial burden in blood and heart from chickens infected with DE142Δorf10 was decreased to 7.2 and 8%, respectively (P < 0.05). DE142Δorf6 showed an obviously attenuated growth rate in the logarithmic phase when cultured in iron-deficient medium, and the transcription level of the iutA gene decreased to 43% (P < 0.05). The bactericidal assays showed that the survival of the mutant DE142Δorf10 was ~60% compared with WT in 50% chicken serum. The K1 capsule-related genes (kpsF, kpsE, kpsC, and kpsM) were down-regulated nearly 2-fold in DE142Δorf10 (P < 0.01). Together, these results suggested that orf6 affects growth by contributing to the uptake ability of iron, while orf10 increases resistance to serum by upregulating K1 capsule-related genes.

Antibiotic Therapy Using Phage Depolymerases: Robustness Across a Range of Conditions

Phage-derived depolymerases directed against bacterial capsules are showing therapeutic promise in various animal models of infection. However, individual animal model studies are often constrained by use of highly specific protocols, such that results may not generalize to even slight modifications. Here we explore the robustness of depolymerase therapies shown to succeed in a previous study of mice. Treatment success rates were reduced by treatment delay, more so for some enzymes than others: K1- and K5 capsule-degrading enzymes retained partial efficacy on delay, while K30 depolymerase did not. Phage were superior to enzymes under delayed treatment only for K1. Route of administration (intramuscular versus intraperitoneal) mattered for success of K1E, possibly for K1F, not for K1H depolymerase. Significantly, K1 capsule-degrading enzymes proved highly successful when using immune-suppressed, leukopenic mice, even with delayed treatment. Evolution of bacteria resistant to K1-degrading enzymes did not thwart therapeutic success in leukopenic mice, likely because resistant bacteria were avirulent. In combination with previous studies these results continue to support the efficacy of depolymerases as antibacterial agents in vivo, but system-specific details are becoming evident.

The Catabolite Repressor Protein-Cyclic AMP Complex RegulatescsgDand Biofilm Formation in Uropathogenic Escherichia coli

ABSTRACTThe extracellular matrix protectsEscherichia colifrom immune cells, oxidative stress, predation, and other environmental stresses. Production of theE. coliextracellular matrix is regulated by transcription factors that are tuned to environmental conditions. The biofilm master regulator protein CsgD upregulates curli and cellulose, the two major polymers in the extracellular matrix of uropathogenicE. coli(UPEC) biofilms. We found that cyclic AMP (cAMP) regulates curli, cellulose, and UPEC biofilms throughcsgD. The alarmone cAMP is produced by adenylate cyclase (CyaA), and deletion ofcyaAresulted in reduced extracellular matrix production and biofilm formation. Thecataboliterepressorprotein (CRP) positively regulatedcsgDtranscription, leading to curli and cellulose production in the UPEC isolate, UTI89. Glucose, a known inhibitor of CyaA activity, blocked extracellular matrix formation when added to the growth medium. The mutant strains ΔcyaAand Δcrpdid not produce rugose biofilms, pellicles, curli, cellulose, or CsgD. Three putative CRP binding sites were identified within thecsgD-csgBintergenic region, and purified CRP could gel shift thecsgD-csgBintergenic region. Additionally, we found that CRP binded upstream ofkpsMT, which encodes machinery for K1 capsule production. Together our work shows that cAMP and CRP influenceE. colibiofilms through transcriptional regulation ofcsgD.IMPORTANCEThecataboliterepressorprotein (CRP)-cyclic AMP (cAMP) complex influences the transcription of ∼7% of genes on theEscherichia colichromosome (D. Zheng, C. Constantinidou, J. L. Hobman, and S. D. Minchin, Nucleic Acids Res 32:5874–5893, 2004,https://dx.doi.org/10.1093/nar/gkh908). Glucose inhibitsE. colibiofilm formation, and ΔcyaAand Δcrpmutants show impaired biofilm formation (D. W. Jackson, J.W. Simecka, and T. Romeo, J Bacteriol 184:3406–3410, 2002,https://dx.doi.org/10.1128/JB.184.12.3406-3410.2002). We determined that the cAMP-CRP complex regulates curli and cellulose production and the formation of rugose and pellicle biofilms throughcsgD. Additionally, we propose that cAMP may work as a signaling compound for uropathogenicE. coli(UPEC) to transition from the bladder lumen to inside epithelial cells for intracellular bacterial community formation through K1 capsule regulation.

The K1 Capsular Polysaccharide from Acinetobacter baumannii Is a Potential Therapeutic Target via Passive Immunization

ABSTRACTThe emergence of extremely resistant and panresistant Gram-negative bacilli, such asAcinetobacter baumannii, requires consideration of nonantimicrobial therapeutic approaches. The goal of this report was to evaluate the K1 capsular polysaccharide fromA. baumanniias a passive immunization target. Its structure was determined by a combination of mass spectrometric and nuclear magnetic resonance (NMR) techniques. Molecular mimics that might raise the concern for autoimmune disease were not identified. Immunization of CD1 mice demonstrated that the K1 capsule is immunogenic. The monoclonal antibody (MAb) 13D6, which is directed against the K1 capsule fromA. baumannii, was used to determine the seroprevalence of the K1 capsule in a collection of 100A. baumanniistrains. Thirteen percent of theA. baumanniiisolates from this collection were seroreactive to MAb 13D6. Opsonization of K1-positive strains, but not K1-negative strains, with MAb 13D6 significantly increased neutrophil-mediated bactericidal activityin vitro(P< 0.05). Lastly, treatment with MAb 13D6 3 and 24 h after bacterial challenge in a rat soft tissue infection model resulted in a significant decrease in the growth/survival of a K1-positive strain compared to that of a K1-negative strain or to treatment with a vehicle control (P< 0.0001). These data support the proof of principle that the K1 capsule is a potential therapeutic target via passive immunization. Other serotypes require assessment, and pragmatic challenges exist, such as the need to serotype infecting strains and utilize serotype-specific therapy. Nonetheless, this approach may become an important therapeutic option with increasing antimicrobial resistance and a diminishing number of active antimicrobials.