Viral Preservation with Protein-Supplemented Nebulizing Media in Aerosols.

The outbreak of SARS-CoV-2 has emphasized the need for a deeper understanding of infectivity, spread, and treatment of airborne viruses. Bacteriophages (phages) serve as ideal surrogates for respiratory pathogenic viruses thanks to their high tractability and the structural similarities tailless phages bear to viral pathogens. However, the aerosolization of enveloped SARS-CoV-2 surrogate phi6 usually results in a >3-log10 reduction in viability, limiting its usefulness as a surrogate for aerosolized coronavirus in "real world" contexts, such as a sneeze or cough. Recent work has shown that saliva or artificial saliva greatly improves the stability of viruses in aerosols and microdroplets relative to standard dilution/storage buffers like suspension medium (SM) buffer. These findings led us to investigate whether we could formulate media that preserves the viability of phi6 and other phages in artificially derived aerosols. Results indicate that SM buffer supplemented with bovine serum albumin (BSA) significantly improves the recovery of airborne phi6, MS2, and 80α and outperforms commercially formulated artificial saliva. Particle sizing and acoustic particle trapping data indicate that BSA supplementation dose-dependently improves viral survivability by reducing the extent of particle evaporation. These data suggest that our viral preservation medium may facilitate a lower-cost alternative to artificial saliva for future applied aerobiology studies. IMPORTANCE We have identified common and inexpensive lab reagents that confer increased aerosol survivability on phi6 and other phages. Our results suggest that soluble protein is a key protective component in nebulizing medium. Protein supplementation likely reduces exposure of the phage to the air-water interface by reducing the extent of particle evaporation. These findings will be useful for applications in which researchers wish to improve the survivability of these (and likely other) aerosolized viruses to better approximate highly transmissible airborne viruses like SARS-CoV-2.

Phage-encoded cationic antimicrobial peptide required for lysis.

Most phages of Gram-negative hosts encode spanins for disruption of the outer membrane, the last step in host lysis. However, bioinformatic analysis indicates that ∼15% of these phages lack a spanin gene, suggesting they have an alternate way of disrupting the OM. Here, we show that the T7-like coliphage phiKT causes the explosive cell lysis associated with spanin activity despite not encoding spanins. A putative lysis cassette cloned from the phiKT late gene region includes the hypothetical novel gene 28 located between the holin and endolysin genes and supports inducible lysis in E. coli K-12. Moreover, induction of an isogenic construct lacking gene 28 resulted in divalent cation-stabilized spherical cells rather than lysis, implicating gp28 in OM disruption. Additionally, gp28 was shown to complement the lysis defect of a spanin-null λ lysogen. Gene 28 encodes a 56-amino acid cationic protein with predicted amphipathic helical structure and is membrane-associated after lysis. Urea and KCl washes did not release gp28 from the particulate, suggesting a strong hydrophobic membrane interaction. Fluorescence microscopy supports membrane localization of the gp28 protein prior to lysis. Gp28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. Synthesized gp28 behaved similar to LL-37 in standard assays mixing peptide and cells to measure bactericidal and inhibitory effects. Taken together, these results indicate that phiKT gp28 is a phage-encoded cationic antimicrobial peptide that disrupts bacterial outer membranes during host lysis and thus establishes a new class of phage lysis proteins, the disruptins. Significance We provide evidence that phiKT produces an antimicrobial peptide for outer membrane disruption during lysis. This protein, designated as a disruptin, is a new paradigm for phage lysis and has no similarities to other known lysis genes. Although many mechanisms have been proposed for the function of antimicrobial peptides, there is no consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic system to support such studies. Therefore, the phiKT disruptin may represent the first genetically tractable antimicrobial peptide, facilitating mechanistic analyses.

Business Data Categorization and Refinement for Application in Longitudinal Neighborhood Health Research: a Methodology.

Retail environments, such as healthcare locations, food stores, and recreation facilities, may be relevant to many health behaviors and outcomes. However, minimal guidance on how to collect, process, aggregate, and link these data results in inconsistent or incomplete measurement that can introduce misclassification bias and limit replication of existing research. We describe the following steps to leverage business data for longitudinal neighborhood health research: re-geolocating establishment addresses, preliminary classification using standard industrial codes, systematic checks to refine classifications, incorporation and integration of complementary data sources, documentation of a flexible hierarchical classification system and variable naming conventions, and linking to neighborhoods and participant residences. We show results of this classification from a dataset of locations (over 77 million establishment locations) across the contiguous U.S. from 1990 to 2014. By incorporating complementary data sources, through manual spot checks in Google StreetView and word and name searches, we enhanced a basic classification using only standard industrial codes. Ultimately, providing these enhanced longitudinal data and supplying detailed methods for researchers to replicate our work promotes consistency, replicability, and new opportunities in neighborhood health research.

Phage spanins: diversity, topological dynamics and gene convergence.

BACKGROUND: Spanins are phage lysis proteins required to disrupt the outer membrane. Phages employ either two-component spanins or unimolecular spanins in this final step of Gram-negative host lysis. Two-component spanins like Rz-Rz1 from phage lambda consist of an integral inner membrane protein: i-spanin, and an outer membrane lipoprotein: o-spanin, that form a complex spanning the periplasm. Two-component spanins exist in three different genetic architectures; embedded, overlapped and separated. In contrast, the unimolecular spanins, like gp11 from phage T1, have an N-terminal lipoylation signal sequence and a C-terminal transmembrane domain to account for the topology requirements. Our proposed model for spanin function, for both spanin types, follows a common theme of the outer membrane getting fused with the inner membrane, effecting the release of progeny virions. RESULTS: Here we present a SpaninDataBase which consists of 528 two-component spanins and 58 unimolecular spanins identified in this analysis. Primary analysis revealed significant differences in the secondary structure predictions for the periplasmic domains of the two-component and unimolecular spanin types, as well as within the three different genetic architectures of the two-component spanins. Using a threshold of 40% sequence identity over 40% sequence length, we were able to group the spanins into 143 i-spanin, 125 o-spanin and 13 u-spanin families. More than 40% of these families from each type were singletons, underlining the extreme diversity of this class of lysis proteins. Multiple sequence alignments of periplasmic domains demonstrated conserved secondary structure patterns and domain organization within family members. Furthermore, analysis of families with members from different architecture allowed us to interpret the evolutionary dynamics of spanin gene arrangement. Also, the potential universal role of intermolecular disulfide bonds in two-component spanin function was substantiated through bioinformatic and genetic approaches. Additionally, a novel lipobox motif, AWAC, was identified and experimentally verified. CONCLUSIONS: The findings from this bioinformatic approach gave us instructive insights into spanin function, evolution, domain organization and provide a platform for future spanin annotation, as well as biochemical and genetic experiments. They also establish that spanins, like viral membrane fusion proteins, adopt different strategies to achieve fusion of the inner and outer membranes.

Suppressor Analysis of the Fusogenic Lambda Spanins.

The final step of lysis in phage λ infections of Escherichia coli is mediated by the spanins Rz and Rz1. These proteins form a complex that bridges the cell envelope and that has been proposed to cause fusion of the inner and outer membranes. Accordingly, mutations that block spanin function are found within coiled-coil domains and the proline-rich region, motifs essential in other fusion systems. To gain insight into spanin function, pseudorevertant alleles that restored plaque formation for lysis-defective mutants of Rz and Rz1 were selected. Most second-site suppressors clustered within a coiled-coil domain of Rz near the outer leaflet of the cytoplasmic membrane and were not allele specific. Suppressors largely encoded polar insertions within the hydrophobic core of the coiled-coil interface. Such suppressor changes resulted in decreased proteolytic stability of the Rz double mutants in vivo Unlike the wild type, in which lysis occurs while the cells retain a rod shape, revertant alleles with second-site suppressor mutations supported lysis events that were preceded by spherical cell formation. This suggests that destabilization of the membrane-proximal coiled coil restores function for defective spanin alleles by increasing the conformational freedom of the complex at the cost of its normal, all-or-nothing functionality.IMPORTANCECaudovirales encode cell envelope-spanning proteins called spanins, which are thought to fuse the inner and outer membranes during phage lysis. Recent genetic analysis identified the functional domains of the lambda spanins, which are similar to class I viral fusion proteins. While the pre- and postfusion structures of model fusion systems have been well characterized, the intermediate structure(s) formed during the fusion reaction remains elusive. Genetic analysis would be expected to identify functional connections between intermediates. Since most membrane fusion systems are not genetically tractable, only few such investigations have been reported. Here, we report a suppressor analysis of lambda spanin function. To our knowledge this is the first suppression analysis of a class I-like complex and also the first such analysis of a prokaryote membrane fusion system.

Membrane fusion during phage lysis.

In general, phages cause lysis of the bacterial host to effect release of the progeny virions. Until recently, it was thought that degradation of the peptidoglycan (PG) was necessary and sufficient for osmotic bursting of the cell. Recently, we have shown that in Gram-negative hosts, phage lysis also requires the disruption of the outer membrane (OM). This is accomplished by spanins, which are phage-encoded proteins that connect the cytoplasmic membrane (inner membrane, IM) and the OM. The mechanism by which the spanins destroy the OM is unknown. Here we show that the spanins of the paradigm coliphage lambda mediate efficient membrane fusion. This supports the notion that the last step of lysis is the fusion of the IM and OM. Moreover, data are provided indicating that spanin-mediated fusion is regulated by the meshwork of the PG, thus coupling fusion to murein degradation by the phage endolysin. Because endolysin function requires the formation of µm-scale holes by the phage holin, the lysis pathway is seen to require dramatic dynamics on the part of the OM and IM, as well as destruction of the PG.

Spatial and temporal control of lysis by the lambda holin.

The infection cycle of phage λ terminates in lysis mediated by three types of lysis proteins, each disrupting a layer in the bacterial envelope: the S105 holin, the R endolysin, and the Rz/Rz1 spanin complex targeting the inner membrane, cell wall or peptidoglycan, and the outer membrane, respectively. Video microscopy has shown that in most infections, lysis occurs as a sudden, explosive event at a cell pole, such that the initial product is a less refractile ghost that retains rod-shaped morphology. Here, we investigate the molecular basis of polar lysis using time-lapse fluorescence microscopy. The results indicate that the holin determines the morphology of lysis by suddenly forming two-dimensional rafts at the poles about 100 s prior to lysis. Given the physiological and biochemical similarities between the lambda holin and other class I holins, dynamic redistribution and sudden concentration may be common features of holins, probably reflecting the fitness advantage of all-or-nothing lysis regulation.IMPORTANCEIn this study, we use fluorescent video microscopy to track -green fluorescent protein (GFP)-labeled holin in the minutes prior to phage lysis. Our work contextualizes prior genetic and biochemical data, showing when hole formation starts and where holin oligomers form in relation to the site of lytic rupture. Furthermore, prior work showed that the morphology of lambda-infected cells is characterized by an explosive event starting at the cell pole; however, the basis for this was not clear. This study shows that holin most often oligomerizes at cell poles and that the site of the oligomerization is spatially correlated with the site of lytic blowout. Therefore, the holin is the key contributor to polar lysis morphology for phage lambda.

Biotic countermeasures that rescue Nannochloropsis gaditana from a Bacillus safensis infection.

The natural assemblage of a symbiotic bacterial microbiome (bacteriome) with microalgae in marine ecosystems is now being investigated as a means to increase algal productivity for industry. When algae are grown in open pond settings, biological contamination causes an estimated 30% loss of the algal crop. Therefore, new crop protection strategies that do not disrupt the native algal bacteriome are needed to produce reliable, high-yield algal biomass. Bacteriophages offer an unexplored solution to treat bacterial pathogenicity in algal cultures because they can eliminate a single species without affecting the bacteriome. To address this, we identified a highly virulent pathogen of the microalga Nannochloropsis gaditana, the bacterium Bacillus safensis, and demonstrated rescue of the microalgae from the pathogen using phage. 16S rRNA amplicon sequencing showed that phage treatment did not alter the composition of the bacteriome. It is widely suspected that the algal bacteriome could play a protective role against bacterial pathogens. To test this, we compared the susceptibility of a bacteriome-attenuated N. gaditana culture challenged with B. safensis to a N. gaditana culture carrying a growth-promoting bacteriome. We showed that the loss of the bacteriome increased the susceptibility of N. gaditana to the pathogen. Transplanting the microalgal bacteriome to the bacteriome-attenuated culture reconstituted the protective effect of the bacteriome. Finally, the success of phage treatment was dependent on the presence of beneficial bacteriome. This study introduces two synergistic countermeasures against bacterial pathogenicity in algal cultures and a tractable model for studying interactions between microalgae, phages, pathogens, and the algae microbiome.

Endolysin Regulation in Phage Mu Lysis.

Bacteriophage Mu is a paradigm coliphage studied mainly because of its use of transposition for genome replication. However, in extensive nonsense mutant screens, only one lysis gene has been identified, the endolysin gp22. This is surprising because in Gram-negative hosts, lysis by Caudovirales phages has been shown to require proteins which disrupt all three layers of the cell envelope. Usually this involves a holin, an endolysin, and a spanin targeting the cytoplasmic membrane, peptidoglycan (PG), and outer membrane (OM), respectively, with the holin determining the timing of lysis initiation. Here, we demonstrate that gp22 is a signal-anchor-release (SAR) endolysin and identify gp23 and gp23.1 as two-component spanin subunits. However, we find that Mu lacks a holin and instead encodes a membrane-tethered cytoplasmic protein, gp25, which is required for the release of the SAR endolysin. Mutational analysis showed that this dependence on gp25 is conferred by lysine residues at positions 6 and 7 of the short cytoplasmic domain of gp22. gp25, which we designate as a releasin, also facilitates the release of SAR endolysins from other phages. Moreover, the entire length of gp25, including its N-terminal transmembrane domain, belongs to a protein family, DUF2730, found in many Mu-like phages, including those with cytoplasmic endolysins. These results are discussed in terms of models for the evolution and mechanism of releasin function and a rationale for Mu lysis without holin control. IMPORTANCE Host cell lysis is the terminal event of the bacteriophage infection cycle. In Gram-negative hosts, lysis requires proteins that disrupt each of the three cell envelope components, only one of which has been identified in Mu: the endolysin gp22. We show that gp22 can be characterized as a SAR endolysin, a muralytic enzyme that activates upon release from the membrane to degrade the cell wall. Furthermore, we identify genes 23 and 23.1 as spanin subunits used for outer membrane disruption. Significantly, we demonstrate that Mu is the first known Caudovirales phage to lack a holin, a protein that disrupts the inner membrane and is traditionally known to release endolysins. In its stead, we report the discovery of a lysis protein, termed the releasin, which Mu uses for SAR endolysin release. This is an example of a system where the dynamic membrane localization of one protein is controlled by a secondary protein.

Revisiting the Effects of Xenon on Urate Oxidase and Tissue Plasminogen Activator: No Evidence for Inhibition by Noble Gases.

Although chemically inert, Xe and other noble gases have been shown to have functional effects on biological systems. For example, Xe is a powerful anesthetic with neuroprotective properties. Recent reports have claimed that Xe inhibits the activity of tissue plasminogen activator (tPA) and urate oxidase (UOX), indicating that the use of Xe as an anesthetic may have undesirable side effects. Here, we revisited the methods used to demonstrate Xe inhibition of UOX and tPA, testing both indirect and direct gas delivery methods with variable bubble sizes and gas flowrates. Our results indicate that Xe or Kr do not affect the activity of UOX or tPA and that the previously reported inhibition is due to protein damage attendant to directly bubbling gases into protein solutions. The lack of evidence to support Xe inhibition of UOX or tPA alleviates concerns regarding possible side effects for the clinical application of Xe as an anesthetic. Furthermore, this study illustrates the importance of using indirect methods of gas dissolution for studying gas-protein interactions in aqueous solution.

Genome-wide screens reveal Escherichia coli genes required for growth of T1-like phage LL5 and V5-like phage LL12.

The host factor requirements of phages and mechanisms of mutational phage insensitivity must be characterized for rational design of phage cocktails. To characterize host dependencies of two novel Escherichia coli phages, the T1-like siphophage LL5 and the V5-like myophage LL12, forward genetic screens were conducted against the Keio collection, a library of single non-essential gene deletions in E. coli str. BW25113. These screens and subsequent experiments identified genes required by phages LL5 and LL12. E. coli mutants deficient in heptose II and the phosphoryl substituent of heptose I of the inner core lipopolysaccharide (LPS) were unable to propagate phage LL5, as were mutants deficient in the outer membrane protein TolC. Mutants lacking glucose I of the LPS outer core failed to propagate LL12. Two additional genes encoding cytoplasmic chaperones, PpiB and SecB, were found to be required for efficient propagation of phage LL5, but not LL12. This screening approach may be useful for identifying host factors dependencies of phages, which would provide valuable information for their potential use as therapeutics and for phage engineering.

Patterns in Geographic Access to Health Care Facilities Across Neighborhoods in the United States Based on Data From the National Establishment Time-Series Between 2000 and 2014.

Importance: The association between proximity to health care facilities and improved disease management and population health has been documented, but little is known about small-area health care environments and how the presence of health care facilities has changed over time during recent health system and policy change. Objective: To examine geographic access to health care facilities across neighborhoods in the United States over a 15-year period. Design, Setting, and Participants: Using longitudinal business data from the National Establishment Time-Series, this cross-sectional study examined the presence of and change in ambulatory care facilities and pharmacies and drugstores in census tracts (CTs) throughout the continental United States between 2000 and 2014. Between January and April 2019, multinomial logistic regression was used to estimate associations between health care facility presence and neighborhood sociodemographic characteristics over time. Main Outcomes and Measures: Change in health care facility presence was measured as never present, lost, gained, or always present between 2000 and 2014. Neighborhood sociodemographic characteristics (ie, CTs) and their change over time were measured from US Census reports (2000 and 2010) and the American Community Survey (2008-2012). Results: Among 72 246 included CTs, the percentage of non-US-born residents, residents 75 years or older, poverty status, and population density increased, and 8.1% of CTs showed a change in the racial/ethnic composition of an area from predominantly non-Hispanic (NH) white to other racial/ethnic composition categories between 2000 and 2010. The presence of ambulatory care facilities increased from a mean (SD) of 7.7 (15.9) per CT in 2000 to 13.0 (22.9) per CT in 2014, and the presence of pharmacies and drugstores increased from a mean (SD) of 0.6 (1.0) per CT in 2000 to 0.9 (1.4) per CT in 2014. Census tracts with predominantly NH black individuals (adjusted odds ratio [aOR], 2.37; 95% CI, 2.03-2.77), Hispanic/Latino individuals (aOR 1.30; 95% CI, 1.00-1.69), and racially/ethnically mixed individuals (aOR, 1.53; 95% CI, 1.33-1.77) in 2000 had higher odds of losing health care facilities between 2000 and 2014 compared with CTs with predominantly NH white individuals, after controlling for other neighborhood characteristics. Census tracts of geographic areas with higher levels of poverty in 2000 also had higher odds of losing health care facilities between 2000 and 2014 (aOR, 1.12; 95% CI, 1.05-1.19). Conclusions and Relevance: Differential change was found in the presence of health care facilities across neighborhoods over time, indicating the need to monitor and address the spatial distribution of health care resources within the context of population health disparities.

Phage Lysis: Multiple Genes for Multiple Barriers.

The first steps in phage lysis involve a temporally controlled permeabilization of the cytoplasmic membrane followed by enzymatic degradation of the peptidoglycan. For Caudovirales of Gram-negative hosts, there are two different systems: the holin-endolysin and pinholin-SAR endolysin pathways. In the former, lysis is initiated when the holin forms micron-scale holes in the inner membrane, releasing active endolysin into the periplasm to degrade the peptidoglycan. In the latter, lysis begins when the pinholin causes depolarization of the membrane, which activates the secreted SAR endolysin. Historically, the disruption of the first two barriers of the cell envelope was thought to be necessary and sufficient for lysis of Gram-negative hosts. However, recently a third functional class of lysis proteins, the spanins, has been shown to be required for outer membrane disruption. Spanins are so named because they form a protein bridge that connects both membranes. Most phages produce a two-component spanin complex, composed of an outer membrane lipoprotein (o-spanin) and an inner membrane protein (i-spanin) with a predominantly coiled-coil periplasmic domain. Some phages have a different type of spanin which spans the periplasm as a single molecule, by virtue of an N-terminal lipoprotein signal and a C-terminal transmembrane domain. Evidence is reviewed supporting a model in which the spanins function by fusing the inner membrane and outer membrane. Moreover, it is proposed that spanin function is inhibited by the meshwork of the peptidoglycan, thus coupling the spanin step to the first two steps mediated by the holin and endolysin.

Complete Genome Sequence of Serratia marcescens Podophage Parlo.

Serratia marcescens is an opportunistic human pathogen with multiple resistance mechanisms that infects hospitalized patients. Here, we report the full genome sequence of S. marcescens podophage Parlo. Parlo is most similar to Erwinia phage PEp14 and encodes a 3,764-residue protein assumed to be a homolog of DarB, an antirestriction protein.

Complete Genome Sequence of Serratia marcescens Phage MTx.

Serratia marcescens is a nosocomial pathogen that has evolved resistance to multiple antibiotics. Here, we present the genome sequence of myophage MTx that infects S. marcescens MTx encodes 103 proteins, with 26 being assigned a predicted function or superfamily classification, and it has little similarity with other phages at the nucleotide level.

Complete Genome Sequence of Serratia marcescens Myophage MyoSmar.

Serratia marcescens is an opportunistic pathogen that causes respiratory, urinary, and digestive tract infections in humans. Here, we describe the annotation of Serratia marcescens myophage MyoSmar. The 68,745-bp genome encodes 105 predicted proteins and is most similar to the genomes of Pseudomonas PB1-like viruses.

Complete Genome Sequence of Escherichia coli Myophage Mangalitsa.

Enteropathogenic Escherichia coli is a prevalent Gram-negative bacterium that can lead to fatal complications from infection in humans. Here, we present the isolation and complete annotation of the 52,329-bp genome of enteropathogenic E. coli ATCC 23545 myophage Mangalitsa. Predicted terminal repeats and temperature sensitivity for plaque formation place Mangalitsa with similar unclassified myoviruses.

Complete Genome Sequence of Staphylococcus aureus Myophage Maine.

Multidrug-resistant strains of Staphylococcus aureus cause serious human disease worldwide. Bacteriophages offer a promising alternative to traditional antibiotics. Here, we announce the 141,712-bp genome of S. aureus phage Maine. A myophage with 9,019-bp predicted terminal repeats and high similarity to other Staphylococcus phages, Maine falls into the Twort-like group.

Genetic Analysis of the Lambda Spanins Rz and Rz1: Identification of Functional Domains.

Coliphage lambda proteins Rz and Rz1 are the inner membrane and outer membrane subunits of the spanin complex-a heterotetramer that bridges the periplasm and is essential for the disruption of the outer membrane during phage lysis. Recent evidence suggests the spanin complex functions by fusing the inner and outer membrane. Here, we use a genetics approach to investigate and characterize determinants of spanin function. Because Rz1 is entirely embedded in the +1 reading frame of Rz, the genes were disembedded before using random mutagenesis to construct a library of lysis-defective alleles for both genes. Surprisingly, most of the lysis-defective missense mutants exhibited normal accumulation or localization in vivo, and also were found to be normal for complex formation in vitro Analysis of the distribution and nature of single missense mutations revealed subdomains that resemble key motifs in established membrane-fusion systems, i.e., two coiled-coil domains in Rz, a proline-rich region of Rz1, and flexible linkers in both proteins. When coding sequences are aligned respective to the embedded genetic architecture of Rz1 within Rz, genetically silent domains of Rz1 correspond to mutationally sensitive domains in Rz, and vice versa, suggesting that the modular structure of the two subunits facilitated the evolutionary compression that resulted in the unique embedded gene architecture.

Complete Genome Sequence of Caulobacter crescentus Podophage Percy.

Podophage Percy infects Caulobacter crescentus, a Gram-negative bacterium that divides asymmetrically and is a commonly used model organism to study the cell cycle, asymmetric cell division, and cell differentiation. Here, we announce the sequence and annotated complete genome of the phiKMV-like podophage Percy and note its prominent features.