High-Throughput Screening of Heterologous Functional Amyloids Using Escherichia coli.

Escherichia coli remains one of the most widely used workhorse microorganisms for the expression of heterologous proteins. The large number of cloning vectors and mutant host strains available for E. coli yields an impressively wide array of folded globular proteins in the laboratory. However, applying modern functional screening approaches to interrogate insoluble protein aggregates such as amyloids requires the use of nonstandard expression pathways. In this chapter, we detail the use of the curli export pathway in E. coli to express a library of gene fragments and variants of a functional amyloid protein to screen sequence traits responsible for aggregation and the formation of nanoscale materials.

A Sarcina bacterium linked to lethal disease in sanctuary chimpanzees in Sierra Leone.

Human and animal infections with bacteria of the genus Sarcina (family Clostridiaceae) are associated with gastric dilation and emphysematous gastritis. However, the potential roles of sarcinae as commensals or pathogens remain unclear. Here, we investigate a lethal disease of unknown etiology that affects sanctuary chimpanzees (Pan troglodytes verus) in Sierra Leone. The disease, which we have named "epizootic neurologic and gastroenteric syndrome" (ENGS), is characterized by neurologic and gastrointestinal signs and results in death of the animals, even after medical treatment. Using a case-control study design, we show that ENGS is strongly associated with Sarcina infection. The microorganism is distinct from Sarcina ventriculi and other known members of its genus, based on bacterial morphology and growth characteristics. Whole-genome sequencing confirms this distinction and reveals the presence of genetic features that may account for the unusual virulence of the bacterium. Therefore, we propose that this organism be considered the representative of a new species, named "Candidatus Sarcina troglodytae". Our results suggest that a heretofore unrecognized complex of related sarcinae likely exists, some of which may be highly virulent. However, the potential role of "Ca. S. troglodytae" in the etiology of ENGS, alone or in combination with other factors, remains a topic for future research.

Engineered Escherichia coli Biofilms Produce Adhesive Nanomaterials Shaped by a Patterned 43 kDa Barnacle Cement Protein.

Barnacles integrate multiple protein components into distinct amyloid-like nanofibers arranged as a bulk material network for their permanent underwater attachment. The design principle for how chemistry is displayed using adhesive nanomaterials, and fragments of proteins that are responsible for their formation, remains a challenge to assess and is yet to be established. Here, we use engineered bacterial biofilms to display a library of amyloid materials outside of the cell using full-length and subdomain sequences from a major component of the barnacle adhesive. A staggered charged pattern is found throughout the full-length sequence of a 43 kDa cement protein (AACP43), establishing a conserved sequence design evolved by barnacles to make adhesive nanomaterials. AACP43 domain deletions vary in their propensity to aggregate and form fibers, as exported extracellular materials are characterized through staining, immunoblotting, scanning electron microscopy, and atomic force microscopy. Full-length AACP43 and its domains have a propensity to aggregate into nanofibers independent of all other barnacle glue components, shedding light on its function in the barnacle adhesive. Curliated Escherichia coli biofilms are a compatible system for heterologous expression and the study of foreign functional amyloid adhesive materials, used here to identify the c-terminal portion of AACP43 as critical in material formation. This approach allows us to establish a common sequence pattern between two otherwise dissimilar families of cement proteins, laying the foundation to elucidate adhesive chemistries by one of the most tenacious marine fouling organisms in the ocean.

Phage-Mediated Molecular Detection (PMMD): A Novel Rapid Method for Phage-Specific Bacterial Detection.

Bacterial infections pose a challenge to human health and burden the health care system, especially with the spread of antibiotic-resistant populations. To provide effective treatment and improved prognosis, effective diagnostic methods are of great importance. Here we present phage-mediated molecular detection (PMMD) as a novel molecular method for the detection and assessment of bacterial antibiotic resistance. This technique consists of a brief incubation, of approximately ten minutes, of the biological sample with a natural bacteriophage (phage) targeting the bacteria of interest. This is followed by total RNA extraction and RT-PCR. We applied this approach to Staphylococcus aureus (SA), a major causative agent of human bacterial infections. PMMD demonstrated a high sensitivity, rapid implementation, and specificity dependent on the phage host range. Moreover, due to the dependence of the signal on the physiological state of the bacteria, PMMD can discriminate methicillin-sensitive from methicillin-resistant SA (MSSA vs. MRSA). Finally, we extended this method to the detection and antibiotic sensitivity determination of other bacteria by proving PMMD efficacy for Bacillusanthracis.

Molecular Recognition of Structures Is Key in the Polymerization of Patterned Barnacle Adhesive Sequences.

The permanent adhesive produced by adult barnacles is held together by tightly folded proteins that form amyloid-like materials distinct among marine foulants. In this work, we link stretches of alternating charged and noncharged linear sequences from a family of adhesive proteins to their role in forming fibrillar nanomaterials. Using recombinant proteins and short barnacle cement derived peptides (BCPs), we find a central sequence with charged motifs of the pattern [Gly/Ser/Val/Thr/Ala-X], where X are charged amino acids, to exert specific control over timing, structure, and morphology of fibril formation. While most BCPs remain dormant, the core segment demonstrates rapid polymerization as well as an ability to template other peptides with no propensity for self-assembly. Patterned charge domains assemble dormant peptides through a specific antiparallel ß-sheet structure as measured by FTIR. While charged domains favor an antiparallel structure, BCPs without charged domains switch fibril assembly to favor simpler parallel ß-sheet aggregates. In addition to activation, charged domains direct nanofibers to grow into discrete microns long fibrils similar to the natural adhesive, while segments without such domains only form short branched aggregates. The assembly of adhesive sequences through recognition of structured templates outlines a strategy used by barnacles to control physical mechanisms of underwater adhesive delivery, activation, and curing based on molecular recognition between proteins.

Development and Use of Personalized Bacteriophage-Based Therapeutic Cocktails To Treat a Patient with a Disseminated Resistant Acinetobacter baumannii Infection.

Widespread antibiotic use in clinical medicine and the livestock industry has contributed to the global spread of multidrug-resistant (MDR) bacterial pathogens, including Acinetobacter baumannii We report on a method used to produce a personalized bacteriophage-based therapeutic treatment for a 68-year-old diabetic patient with necrotizing pancreatitis complicated by an MDR A. baumannii infection. Despite multiple antibiotic courses and efforts at percutaneous drainage of a pancreatic pseudocyst, the patient deteriorated over a 4-month period. In the absence of effective antibiotics, two laboratories identified nine different bacteriophages with lytic activity for an A. baumannii isolate from the patient. Administration of these bacteriophages intravenously and percutaneously into the abscess cavities was associated with reversal of the patient's downward clinical trajectory, clearance of the A. baumannii infection, and a return to health. The outcome of this case suggests that the methods described here for the production of bacteriophage therapeutics could be applied to similar cases and that more concerted efforts to investigate the use of therapeutic bacteriophages for MDR bacterial infections are warranted.

Characterization of novel Staphylococcus aureus lytic phage and defining their combinatorial virulence using the OmniLog® system.

Skin and soft tissue infections (SSTI) caused by methicillin resistant Staphylococcus aureus (MRSA) are difficult to treat. Bacteriophage (phage) represent a potential alternate treatment for antibiotic resistant bacterial infections. In this study, 7 novel phage with broad lytic activity for S. aureus were isolated and identified. Screening of a diverse collection of 170 clinical isolates by efficiency of plating (EOP) assays shows that the novel phage are virulent and effectively prevent growth of 70-91% of MRSA and methicillin sensitive S. aureus (MSSA) isolates. Phage K, which was previously identified as having lytic activity on S. aureus was tested on the S. aureus collection and shown to prevent growth of 82% of the isolates. These novel phage group were examined by electron microscopy, the results of which indicate that the phage belong to the Myoviridae family of viruses. The novel phage group requires ß-N-acetyl glucosamine (GlcNac) moieties on cell wall teichoic acids for infection. The phage were distinct from, but closely related to, phage K as characterized by restriction endonuclease analysis. Furthermore, growth rate analysis via OmniLog® microplate assay indicates that a combination of phage K, with phage SA0420ᶲ1, SA0456ᶲ1 or SA0482ᶲ1 have a synergistic phage-mediated lytic effect on MRSA and suppress formation of phage resistance. These results indicate that a broad spectrum lytic phage mixture can suppress the emergence of resistant bacterial populations and hence have great potential for combating S. aureus wound infections.

The role of HuR in the post-transcriptional regulation of interleukin-3 in T cells.

Human Interleukin-3 (IL-3) is a lymphokine member of a class of transiently expressed mRNAs harboring Adenosine/Uridine-Rich Elements (ARE) in their 3' untranslated regions (3'-UTRs). The regulatory effects of AREs are often mediated by specific ARE-binding proteins (ARE-BPs). In this report, we show that the human IL-3 3'-UTR plays a post-transcriptional regulation role in two human transformed cell lines. More specifically, we demonstrate that the hIL-3 3'-UTR represses the translation of a luciferase reporter both in HeLa and Jurkat T-cells. These results also revealed that the hIL-3 3'-UTR-mediated translational repression is exerted by an 83 nt region comprised mainly by AREs and some non-ARE sequences. Moreover, electrophoretic mobility shift assays (EMSAs) and UV-crosslinking analysis show that this hIL-3 ARE-rich region recruits five specific protein complexes, including the ARE-BPs HuR and TIA-1. HuR binding to this ARE-rich region appears to be spatially modulated during T-cell activation. Together, these results suggest that HuR recognizes the ARE-rich region and plays a role in the IL-3 3'-UTR-mediated post-transcriptional control in T-cells.

Identification and functional analysis of novel phosphorylation sites in the RNA surveillance protein Upf1.

One third of inherited genetic diseases are caused by mRNAs harboring premature termination codons as a result of nonsense mutations. These aberrant mRNAs are degraded by the Nonsense-Mediated mRNA Decay (NMD) pathway. A central component of the NMD pathway is Upf1, an RNA-dependent ATPase and helicase. Upf1 is a known phosphorylated protein, but only portions of this large protein have been examined for phosphorylation sites and the functional relevance of its phosphorylation has not been elucidated in Saccharomyces cerevisiae. Using tandem mass spectrometry analyses, we report the identification of 11 putative phosphorylated sites in S. cerevisiae Upf1. Five of these phosphorylated residues are located within the ATPase and helicase domains and are conserved in higher eukaryotes, suggesting a biological significance for their phosphorylation. Indeed, functional analysis demonstrated that a small carboxy-terminal motif harboring at least three phosphorylated amino acids is important for three Upf1 functions: ATPase activity, NMD activity and the ability to promote translation termination efficiency. We provide evidence that two tyrosines within this phospho-motif (Y-738 and Y-742) act redundantly to promote ATP hydrolysis, NMD efficiency and translation termination fidelity.

The shuttling protein Npl3 promotes translation termination accuracy in Saccharomyces cerevisiae.

Heterogeneous nuclear ribonucleoproteins are multifunctional proteins that bind to newly synthesized mRNAs in the nucleus and participate in many subsequent steps of gene expression. A well-studied Saccharomyces cerevisiae heterogeneous nuclear ribonucleoprotein that has several nuclear functions is Npl3p. Here, we provide evidence that Npl3p also has a cytoplasmic role: it functions in translation termination fidelity. Yeast harboring the npl3-95 mutant allele have an impaired ability to translate lacZ, enhanced sensitivity to cycloheximide and paromomycin, and increased ability to read through translation termination codons. Most of these defects are enhanced in yeast that also lack Upf1p, an RNA surveillance factor crucial for translation termination. We show that the npl3-95 mutant allele encodes a form of Npl3p that is part of high molecular-weight complexes that cofractionate with the poly(A)-binding protein Pab1p. Together, these results lead us to propose a model in which Npl3p engenders translational fidelity by promoting the remodeling of mRNPs during translation termination.

The Rsp5 E3 ligase mediates turnover of low affinity phosphate transporters in Saccharomyces cerevisiae.

In an effort to identify novel components of the PHO regulon in Saccharomyces cerevisiae, we have isolated and characterized suppressors of the Pho(-) phenotype associated with deletion of the Pho4 transcriptional activator. Here we report that either a defective form of the Rsp5 E3 ubiquitin ligase or deletion of the End3 component of the endocytic pathway restores growth of the pho4 Delta mutant in the presence of limiting inorganic phosphate (P i). The spa1-1 suppressor allele of RSP5 encodes a phenylalanine-to-valine replacement at position 748 (F748V) within the catalytic HECT domain of Rsp5. Consistent with suppression due to impaired ubiquitin ligase activity, the heat-sensitive growth defect of the spa1-1 mutant is suppressed either by overexpression of ubiquitin or by osmotic stabilization. Western blot analyses revealed that the cellular levels of the Pho87 and Pho91 low affinity P i are markedly increased in the spa1-1 mutant, yet Pho84 high affinity P i transporter levels are unaffected. Furthermore, Pho87 and Pho91 are ubiquitinated in vivo in an Rsp5-dependent manner, and the Pho+ phenotype of the spa1-1 suppressor is dependent upon Pho87 and Pho91. We conclude that turnover of the low affinity P i transporters is initiated by Rsp5-mediated ubiquitination followed by internalization and degradation by the endocytic pathway.