HSP90 is part of a protein complex with the L polymerase of Rift Valley fever phlebovirus and prevents its degradation by the proteasome during the viral genome replication/transcription stage.

The mosquito-borne Rift Valley fever virus (RVFV) from the Phenuiviridae family is a single-stranded RNA virus that causes the re-emerging zoonotic disease Rift Valley fever (RVF). Classified as a Category A agent by the NIH, RVFV infection can cause debilitating disease or death in humans and lead to devastating economic impacts by causing abortion storms in pregnant cattle. In a previous study, we showed that the host chaperone protein HSP90 is an RVFV-associated host factor that plays a critical role post viral entry, during the active phase of viral genome replication/transcription. In this study, we have elucidated the molecular mechanisms behind the regulatory effect of HSP90 during infection with RVFV. Our results demonstrate that during the early infection phase, host HSP90 associates with the viral RNA-dependent RNA polymerase (L protein) and prevents its degradation through the proteasome, resulting in increased viral replication.

Gravity-perfused airway-on-a-chip optimized for quantitative BSL-3 studies of SARS-CoV-2 infection: barrier permeability, cytokine production, immunohistochemistry, and viral load assays.

Human microphysiological systems, such as organs on chips, are an emerging technology for modeling human physiology in a preclinical setting to understand the mechanism of action of drugs, to evaluate the efficacy of treatment options for human disease and impairment, and to assess drug toxicity. By using human cells co-cultured in three-dimensional constructs, organ chips can provide greater fidelity to the human cellular condition than their two-dimensional predecessors. However, with the rise of SARS-CoV-2 and the global COVID-19 pandemic, it became clear that many microphysiological systems were not compatible with or optimized for studies of infectious disease and operation in a Biosafety Level 3 (BSL-3) environment. Given that one of the early sites of SARS-CoV-2 infection is the airway, we created a human airway organ chip that could operate in a BSL-3 space with high throughput and minimal manipulation, while retaining the necessary physical and physiological components to recapitulate tissue response to infectious agents and the immune response to infection.

Exogenous Klotho Extends Survival in COVID-19 Model Mice.

A striking feature of COVID-19 disease is the broad spectrum of risk factors associated with case severity, as well as the diversity of clinical manifestations. While no central agent has been able to explain the pathogenesis of SARS-CoV-2 infection, the factors that most robustly correlate with severity are risk factors linked to aging. Low serum levels of Klotho, an anti-aging protein, strongly correlate with the pathogenesis of the same risk factors and manifestations of conditions similar to those expressed in severe COVID-19 cases. The current manuscript presents original research on the effects of the exogenous application of Klotho, an anti-aging protein, in COVID-19 model mice. Klotho supplementation resulted in a statistically significant survival benefit in parametric and non-parametric models. Further research is required to elucidate the mechanistic role Klotho plays in COVID-19 pathogenesis as well as the possible modulation SARS-CoV-2 may have on the biological aging process.

Anticancer pan-ErbB inhibitors reduce inflammation and tissue injury and exert broad-spectrum antiviral effects.

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family of receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, ErbB2, and ErbB4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, proinflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production, and disruption of blood-brain barrier integrity in microfluidics-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof of principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

Interactions of Equine Viruses with the Host Kinase Machinery and Implications for One Health and Human Disease.

Zoonotic pathogens that are vector-transmitted have and continue to contribute to several emerging infections globally. In recent years, spillover events of such zoonotic pathogens have increased in frequency as a result of direct contact with livestock, wildlife, and urbanization, forcing animals from their natural habitats. Equines serve as reservoir hosts for vector-transmitted zoonotic viruses that are also capable of infecting humans and causing disease. From a One Health perspective, equine viruses, therefore, pose major concerns for periodic outbreaks globally. Several equine viruses have spread out of their indigenous regions, such as West Nile virus (WNV) and equine encephalitis viruses (EEVs), making them of paramount concern to public health. Viruses have evolved many mechanisms to support the establishment of productive infection and to avoid host defense mechanisms, including promoting or decreasing inflammatory responses and regulating host machinery for protein synthesis. Viral interactions with the host enzymatic machinery, specifically kinases, can support the viral infectious process and downplay innate immune mechanisms, cumulatively leading to a more severe course of the disease. In this review, we will focus on how select equine viruses interact with host kinases to support viral multiplication.

Inhibitors of the Ubiquitin-Mediated Signaling Pathway Exhibit Broad-Spectrum Antiviral Activities against New World Alphaviruses.

New World alphaviruses including Venezuelan Equine Encephalitis Virus (VEEV) and Eastern Equine Encephalitis Virus (EEEV) are mosquito-transmitted viruses that cause disease in humans and equines. There are currently no FDA-approved therapeutics or vaccines to treat or prevent exposure-associated encephalitic disease. The ubiquitin proteasome system (UPS)-associated signaling events are known to play an important role in the establishment of a productive infection for several acutely infectious viruses. The critical engagement of the UPS-associated signaling mechanisms by many viruses as host-pathogen interaction hubs led us to hypothesize that small molecule inhibitors that interfere with these signaling pathways will exert broad-spectrum inhibitory activity against alphaviruses. We queried eight inhibitors of the UPS signaling pathway for antiviral outcomes against VEEV. Three of the tested inhibitors, namely NSC697923 (NSC), bardoxolone methyl (BARM) and omaveloxolone (OMA) demonstrated broad-spectrum antiviral activity against VEEV and EEEV. Dose dependency and time of addition studies suggest that BARM and OMA exhibit intracellular and post-entry viral inhibition. Cumulatively, our studies indicate that inhibitors of the UPS-associated signaling pathways exert broad-spectrum antiviral outcomes in the context of VEEV and EEEV infection, supporting their translational application as therapeutic candidates to treat alphavirus infections.

DNA origami presenting the receptor binding domain of SARS-CoV-2 elicit robust protective immune response.

Effective and safe vaccines are invaluable tools in the arsenal to fight infectious diseases. The rapid spreading of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the coronavirus disease 2019 pandemic has highlighted the need to develop methods for rapid and efficient vaccine development. DNA origami nanoparticles (DNA-NPs) presenting multiple antigens in prescribed nanoscale patterns have recently emerged as a safe, efficient, and easily scalable alternative for rational design of vaccines. Here, we are leveraging the unique properties of these DNA-NPs and demonstrate that precisely patterning ten copies of a reconstituted trimer of the receptor binding domain (RBD) of SARS-CoV-2 along with CpG adjuvants on the DNA-NPs is able to elicit a robust protective immunity against SARS-CoV-2 in a mouse model. Our results demonstrate the potential of our DNA-NP-based approach for developing safe and effective nanovaccines against infectious diseases with prolonged antibody response and effective protection in the context of a viral challenge.

Anticancer pan-ErbB inhibitors reduce inflammation and tissue injury and exert broad-spectrum antiviral effects.

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, 2 and 4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, pro-inflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production and disruption of the blood-brain barrier integrity in microfluidic-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof-of-principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

Brilacidin as a Broad-Spectrum Inhibitor of Enveloped, Acutely Infectious Viruses.

Alphaviruses, belonging to the Togaviridae family, and bunyaviruses, belonging to the Paramyxoviridae family, are globally distributed and lack FDA-approved vaccines and therapeutics. The alphaviruses Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV) are known to cause severe encephalitis, whereas Sindbis virus (SINV) causes arthralgia potentially persisting for years after initial infection. The bunyavirus Rift Valley Fever virus (RVFV) can lead to blindness, liver failure, and hemorrhagic fever. Brilacidin, a small molecule that was designed de novo based on naturally occurring host defensins, was investigated for its antiviral activity against these viruses in human small airway epithelial cells (HSAECs) and African green monkey kidney cells (Veros). This testing was further expanded into a non-enveloped Echovirus, a Picornavirus, to further demonstrate brilacidin's effect on early steps of the viral infectious cycle that leads to inhibition of viral load. Brilacidin demonstrated antiviral activity against alphaviruses VEEV TC-83, VEEV TrD, SINV, EEEV, and bunyavirus RVFV. The inhibitory potential of brilacidin against the viruses tested in this study was dependent on the dosing strategy which necessitated compound addition pre- and post-infection, with addition only at the post-infection stage not eliciting a robust inhibitory response. The inhibitory activity of brilacidin was only modest in the context of the non-enveloped Picornavirus Echovirus, suggesting brilacidin may be less potent against non-enveloped viruses.

Application of a Human Blood Brain Barrier Organ-on-a-Chip Model to Evaluate Small Molecule Effectiveness against Venezuelan Equine Encephalitis Virus.

The blood brain barrier (BBB) is a multicellular microenvironment that plays an important role in regulating bidirectional transport to and from the central nervous system (CNS). Infections by many acutely infectious viruses such as alphaviruses and flaviviruses are known to impact the integrity of the endothelial lining of the BBB. Infection by Venezuelan Equine Encephalitis Virus (VEEV) through the aerosol route causes significant damage to the integrity of the BBB, which contributes to long-term neurological sequelae. An effective therapeutic intervention strategy should ideally not only control viral load in the host, but also prevent and/or reverse deleterious events at the BBB. Two dimensional monocultures, including trans-well models that use endothelial cells, do not recapitulate the intricate multicellular environment of the BBB. Complex in vitro organ-on-a-chip models (OOC) provide a great opportunity to introduce human-like experimental models to understand the mechanistic underpinnings of the disease state and evaluate the effectiveness of therapeutic candidates in a highly relevant manner. Here we demonstrate the utility of a neurovascular unit (NVU) in analyzing the dynamics of infection and proinflammatory response following VEEV infection and therapeutic effectiveness of omaveloxolone to preserve BBB integrity and decrease viral and inflammatory load.

Inhibition of Venezuelan Equine Encephalitis Virus Using Small Interfering RNAs.

Acutely infectious new world alphaviruses such as Venezuelan Equine Encephalitis Virus (VEEV) pose important challenges to the human population due to a lack of effective therapeutic intervention strategies. Small interfering RNAs that can selectively target the viral genome (vsiRNAs) has been observed to offer survival advantages in several in vitro and in vivo models of acute virus infections, including alphaviruses such as Chikungunya virus and filoviruses such as Ebola virus. In this study, novel vsiRNAs that targeted conserved regions in the nonstructural and structural genes of the VEEV genome were designed and evaluated for antiviral activity in mammalian cells in the context of VEEV infection. The data demonstrate that vsiRNAs were able to effectively decrease the infectious virus titer at earlier time points post infection in the context of the attenuated TC-83 strain and the virulent Trinidad Donkey strain, while the inhibition was overcome at later time points. Depletion of Argonaute 2 protein (Ago2), the catalytic component of the RISC complex, negated the inhibitory effect of the vsiRNAs, underscoring the involvement of the siRNA pathway in the inhibition process. Depletion of the RNAi pathway proteins Dicer, MOV10, TRBP2 and Matrin 3 decreased viral load in infected cells, alluding to an impact of the RNAi pathway in the establishment of a productive infection. Additional studies focused on rational combinations of effective vsiRNAs and delivery strategies to confer better in vivo bioavailability and distribution to key target tissues such as the brain can provide effective solutions to treat encephalitic diseases resulting from alphavirus infections.

Development of a hybrid alphavirus-SARS-CoV-2 pseudovirion for rapid quantification of neutralization antibodies and antiviral drugs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S)-pseudotyped viruses are commonly used for quantifying antiviral drugs and neutralizing antibodies. Here, we describe the development of a hybrid alphavirus-SARS-CoV-2 (Ha-CoV-2) pseudovirion, which is a non-replicating SARS-CoV-2 virus-like particle composed of viral structural proteins (S, M, N, and E) and an RNA genome derived from a fast-expressing alphaviral vector. We validated Ha-CoV-2 for rapid quantification of neutralization antibodies, antiviral drugs, and viral variants. In addition, as a proof of concept, we used Ha-CoV-2 to quantify the neutralizing antibodies from an infected and vaccinated individual and found that the one-dose vaccination with Moderna mRNA-1273 greatly increased the anti-serum titer by approximately 6-fold. The post-vaccination serum can neutralize all nine variants tested. These results demonstrate that Ha-CoV-2 can be used as a robust platform for the rapid quantification of neutralizing antibodies against SARS-CoV-2 and its emerging variants.

Maraviroc inhibits SARS-CoV-2 multiplication and s-protein mediated cell fusion in cell culture.

In an effort to identify therapeutic intervention strategies for the treatment of COVID-19, we have investigated a selection of FDA-approved small molecules and biologics that are commonly used to treat other human diseases. A investigation into 18 small molecules and 3 biologics was conducted in cell culture and the impact of treatment on viral titer was quantified by plaque assay. The investigation identified 4 FDA-approved small molecules, Maraviroc, FTY720 (Fingolimod), Atorvastatin and Nitazoxanide that were able to inhibit SARS-CoV-2 infection. Confocal microscopy with over expressed S-protein demonstrated that Maraviroc reduced the extent of S-protein mediated cell fusion as observed by fewer multinucleate cells in the context of drug-treatment. Mathematical modeling of drug-dependent viral multiplication dynamics revealed that prolonged drug treatment will exert an exponential decrease in viral load in a multicellular/tissue environment. Taken together, the data demonstrate that Maraviroc, Fingolimod, Atorvastatin and Nitazoxanide inhibit SARS-CoV-2 in cell culture.

Exosomes originating from infection with the cytoplasmic single-stranded RNA virus Rift Valley fever virus (RVFV) protect recipient cells by inducing RIG-I mediated IFN-B response that leads to activation of autophagy.

BACKGROUND: Although multiple studies have demonstrated a role for exosomes during virus infections, our understanding of the mechanisms by which exosome exchange regulates immune response during viral infections and affects viral pathogenesis is still in its infancy. In particular, very little is known for cytoplasmic single-stranded RNA viruses such as SARS-CoV-2 and Rift Valley fever virus (RVFV). We have used RVFV infection as a model for cytoplasmic single-stranded RNA viruses to address this gap in knowledge. RVFV is a highly pathogenic agent that causes RVF, a zoonotic disease for which no effective therapeutic or approved human vaccine exist. RESULTS: We show here that exosomes released from cells infected with RVFV (designated as EXi-RVFV) serve a protective role for the host and provide a mechanistic model for these effects. Our results show that treatment of both naïve immune cells (U937 monocytes) and naïve non-immune cells (HSAECs) with EXi-RVFV induces a strong RIG-I dependent activation of IFN-B. We also demonstrate that this strong anti-viral response leads to activation of autophagy in treated cells and correlates with resistance to subsequent viral infection. Since we have shown that viral RNA genome is associated with EXi-RVFV, RIG-I activation might be mediated by the presence of packaged viral RNA sequences. CONCLUSIONS: Using RVFV infection as a model for cytoplasmic single-stranded RNA viruses, our results show a novel mechanism of host protection by exosomes released from infected cells (EXi) whereby the EXi activate RIG-I to induce IFN-dependent activation of autophagy in naïve recipient cells including monocytes. Because monocytes serve as reservoirs for RVFV replication, this EXi-RVFV-induced activation of autophagy in monocytes may work to slow down or halt viral dissemination in the infected organism. These findings offer novel mechanistic insights that may aid in future development of effective vaccines or therapeutics, and that may be applicable for a better molecular understanding of how exosome release regulates innate immune response to other cytoplasmic single-stranded RNA viruses.

Inhibitors of Venezuelan Equine Encephalitis Virus Identified Based on Host Interaction Partners of Viral Non-Structural Protein 3.

Venezuelan equine encephalitis virus (VEEV) is a new world alphavirus and a category B select agent. Currently, no FDA-approved vaccines or therapeutics are available to treat VEEV exposure and resultant disease manifestations. The C-terminus of the VEEV non-structural protein 3 (nsP3) facilitates cell-specific and virus-specific host factor binding preferences among alphaviruses, thereby providing targets of interest when designing novel antiviral therapeutics. In this study, we utilized an overexpression construct encoding HA-tagged nsP3 to identify host proteins that interact with VEEV nsP3 by mass spectrometry. Bioinformatic analyses of the putative interactors identified 42 small molecules with the potential to inhibit the host interaction targets, and thus potentially inhibit VEEV. Three inhibitors, tomatidine, citalopram HBr, and Z-VEID-FMK, reduced replication of both the TC-83 strain and the Trinidad donkey (TrD) strain of VEEV by at least 10-fold in astrocytoma, astroglial, and microglial cells. Further, these inhibitors reduced replication of the related New World (NW) alphavirus Eastern equine encephalitis virus (EEEV) in multiple cell types, thus demonstrating broad-spectrum antiviral activity. Time-course assays revealed all three inhibitors reduced both infectious particle production and positive-sense RNA levels post-infection. Further evaluation of the putative host targets for the three inhibitors revealed an interaction of VEEV nsP3 with TFAP2A, but not eIF2S2. Mechanistic studies utilizing siRNA knockdowns demonstrated that eIF2S2, but not TFAP2A, supports both efficient TC-83 replication and genomic RNA synthesis, but not subgenomic RNA translation. Overall, this work reveals the composition of the VEEV nsP3 proteome and the potential to identify host-based, broad spectrum therapeutic approaches to treat new world alphavirus infections.

Durability of Viral Neutralization in Asymptomatic Coronavirus Disease 2019 for at Least 60 Days.

A cohort consisting of asymptomatic healthcare workers donated temporal serum samples after infection with severe acute respiratory syndrome coronavirus 2. Analysis shows that all asymptomatic healthcare workers had neutralizing antibodies, that these antibodies persist for ≥60 days, and that anti-spike receptor-binding domain immunoglobulin G levels were correspondingly durable over the same time period.

Extracellular vesicles from HTLV-1 infected cells modulate target cells and viral spread.

BACKGROUND: The Human T-cell Lymphotropic Virus Type-1 (HTLV-1) is a blood-borne pathogen and etiological agent of Adult T-cell Leukemia/Lymphoma (ATLL) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). HTLV-1 has currently infected up to 10 million globally with highly endemic areas in Japan, Africa, the Caribbean and South America. We have previously shown that Extracellular Vesicles (EVs) enhance HTLV-1 transmission by promoting cell-cell contact. RESULTS: Here, we separated EVs into subpopulations using differential ultracentrifugation (DUC) at speeds of 2 k (2000×g), 10 k (10,000×g), and 100 k (100,000×g) from infected cell supernatants. Proteomic analysis revealed that EVs contain the highest viral/host protein abundance in the 2 k subpopulation (2 k > 10 k > 100 k). The 2 k and 10 k populations contained viral proteins (i.e., p19 and Tax), and autophagy proteins (i.e., LC3 and p62) suggesting presence of autophagosomes as well as core histones. Interestingly, the use of 2 k EVs in an angiogenesis assay (mesenchymal stem cells + endothelial cells) caused deterioration of vascular-like-tubules. Cells commonly associated with the neurovascular unit (i.e., astrocytes, neurons, and macrophages) in the blood-brain barrier (BBB) showed that HTLV-1 EVs may induce expression of cytokines involved in migration (i.e., IL-8; 100 k > 2 k > 10 k) from astrocytes and monocyte-derived macrophages (i.e., IL-8; 2 k > 10 k). Finally, we found that EVs were able to promote cell-cell contact and viral transmission in monocytic cell-derived dendritic cell. The EVs from both 2 k and 10 k increased HTLV-1 spread in a humanized mouse model, as evidenced by an increase in proviral DNA and RNA in the Blood, Lymph Node, and Spleen. CONCLUSIONS: Altogether, these data suggest that various EV subpopulations induce cytokine expression, tissue damage, and viral spread.

Brilacidin Demonstrates Inhibition of SARS-CoV-2 in Cell Culture.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the newly emergent causative agent of coronavirus disease-19 (COVID-19), has resulted in more than two million deaths worldwide since it was first detected in 2019. There is a critical global need for therapeutic intervention strategies that can be deployed to safely treat COVID-19 disease and reduce associated morbidity and mortality. Increasing evidence shows that both natural and synthetic antimicrobial peptides (AMPs), also referred to as Host Defense Proteins/Peptides (HDPs), can inhibit SARS-CoV-2, paving the way for the potential clinical use of these molecules as therapeutic options. In this manuscript, we describe the potent antiviral activity exerted by brilacidin-a de novo designed synthetic small molecule that captures the biological properties of HDPs-on SARS-CoV-2 in a human lung cell line (Calu-3) and a monkey cell line (Vero). These data suggest that SARS-CoV-2 inhibition in these cell culture models is likely to be a result of the impact of brilacidin on viral entry and its disruption of viral integrity. Brilacidin demonstrated synergistic antiviral activity when combined with remdesivir. Collectively, our data demonstrate that brilacidin exerts potent inhibition of SARS-CoV-2 against different strains of the virus in cell culture.

VOC fingerprints: metabolomic signatures of biothreat agents with and without antibiotic resistance.

Category A and B biothreat agents are deemed to be of great concern by the US Centers for Disease Control and Prevention (CDC) and include the bacteria Francisella tularensis, Yersinia pestis, Burkholderia mallei, and Brucella species. Underscored by the impact of the 2020 SARS-CoV-2 outbreak, 2016 Zika pandemic, 2014 Ebola outbreak, 2001 anthrax letter attacks, and 1984 Rajneeshee Salmonella attacks, the threat of future epidemics/pandemics and/or terrorist/criminal use of pathogenic organisms warrants continued exploration and development of both classic and alternative methods of detecting biothreat agents. Volatile organic compounds (VOCs) comprise a large and highly diverse group of carbon-based molecules, generally related by their volatility at ambient temperature. Recently, the diagnostic potential of VOCs has been realized, as correlations between the microbial VOC metabolome and specific bacterial pathogens have been identified. Herein, we describe the use of microbial VOC profiles as fingerprints for the identification of biothreat-relevant microbes, and for differentiating between a kanamycin susceptible and resistant strain. Additionally, we demonstrate microbial VOC profiling using a rapid-throughput VOC metabolomics method we refer to as 'simultaneous multifiber headspace solid-phase microextraction' (simulti-hSPME). Finally, through VOC analysis, we illustrate a rapid non-invasive approach to the diagnosis of BALB/c mice infected with either F. tularensis SCHU S4 or Y. pestis CO92.

Discovery and Biochemical Characterization of PlyP56, PlyN74, and PlyTB40-Bacillus Specific Endolysins.

Three Bacillus bacteriophage-derived endolysins, designated PlyP56, PlyN74, and PlyTB40, were identified, cloned, purified, and characterized for their antimicrobial properties. Sequence alignment reveals these endolysins have an N-terminal enzymatically active domain (EAD) linked to a C-terminal cell wall binding domain (CBD). PlyP56 has a Peptidase_M15_4/VanY superfamily EAD with a conserved metal binding motif and displays biological dependence on divalent ions for activity. In contrast, PlyN74 and PlyTB40 have T7 lysozyme-type Amidase_2 and carboxypeptidase T-type Amidase_3 EADs, respectively, which are members of the MurNAc-LAA superfamily, but are not homologs and thus do not have a shared protein fold. All three endolysins contain similar SH3-family CBDs. Although minor host range differences were noted, all three endolysins show relatively broad antimicrobial activity against members of the Bacillus cereus sensu lato group with the highest lytic activity against B. cereus ATCC 4342. Characterization studies determined the optimal lytic activity for these enzymes was at physiological pH (pH 7.0⁻8.0), over a broad temperature range (4⁻55 °C), and at low concentrations of NaCl (<50 mM). Direct comparison of lytic activity shows the PlyP56 enzyme to be twice as effective at lysing the cell wall peptidoglycan as PlyN74 or PlyTB40, suggesting PlyP56 is a good candidate for further antimicrobial development as well as bioengineering studies.