Biology subjects

Waters, C. M.

Publications and source records attributed to Waters, C. M..

At least 19 recordsLinked to original sources

bioRxiv2025

Phages use contingency loci as a bet-hedging strategy

Bacteriophages are estimated to outnumber bacteria by [~]10-fold1,2. Here, we show that phage genomes contain contingency loci (CL), hypermutable DNA regions that promote reversible frameshift mutations through DNA polymerase slippage3-6. CL have been described in bacteria, archaea, and eukaryotes but have not previously been reported in phages. We demonstrate that CL in coliphage T2 and T4 generates genomic and phenotypic diversity in resulting progeny to evade host defense, a process known as bet-hedging. Whole genome sequencing of T2 and T4 show similar levels of CL-driven sequence variation in dozens of other putative CL. Additional sequencing of T6, T7, Sec{varphi}27, ICP1 and ICP2, alongside bioinformatics of the BASEL phage collection reveals that putative CL are widespread in phages and are encoded in every functional class of genes. Collectively, our study describes a new paradigm for understanding phage replication in which CL drive genetic diversification and population heterogeneity to rapidly evolve.

microbiology↗

bioRxiv2025

Analyzing the role of a dual-function diguanylate cyclase/phosphodiesterase for attachment and motility in Agrobacterium tumefaciens

Cyclic diguanylate monophosphate (c-di-GMP) is a second messenger that controls the signaling pathway for the motile to sessile transition in many bacteria. Diguanylate cyclases (DGCs), characterized by a GGDEF domain, are responsible for the synthesis of c-di-GMP. c-di-GMP is degraded by EAL and HD-GYP domains that exhibit phosphodiesterase activity (PDE). In Agrobacterium tumefaciens, out of 31 predicted proteins regulating the c-di-GMP levels, this work focuses on the predicted dual-function DGC/PDE, DcpB. We show that DcpB is a cycle-dependent PDE under our experimental conditions, resulting in cell cycle-dependent control of motility and biofilm formation in A. tumefaciens. DcpB also exhibits polar and mid-cell localization. Finally, we identify genetic interactions between dcpB and a LacI-family transcriptional regulators, thuR, suggesting additional regulatory inputs for DcpB-dependent phenotypes. Importancec-di-GMP is a universal secondary messenger known to control various biological processes in bacteria including attachment, motility, virulence, and cell cycle progression. In A. tumefaciens, there are 31 predicted c-di-GMP-metabolizing proteins predicted to affect the c-di-GMP pool, raising the question of why so many proteins are involved and how the activities of these proteins are coordinated. This work describes a cell cycle-dependent PDE activity that helps coordinate cell cycle progression and developmental phenotypes including biofilm formation and motility.

microbiology↗

bioRxiv2025

A Phage Ejectosome Protein Moonlights to Inhibit CBASS Anti-Viral Defense System

A family of prokaryotic antiviral systems collectively known as the cyclic oligonucleotide-based signaling system (CBASS) is found in many bacterial species. Phage defense in CBASS is mediated by the action of a specific effector activated by the cognate cyclic oligonucleotide, often resulting in abortive infection. In the prototypical Type II-A CBASS, phage defense is initiated by the production of cyclic GMP-AMP (cGAMP) by enzymes known as cGAS/DncV-like nucleotidyltransferases (CD-NTases) within the bacterial host, and subsequent activation of the CapV phospholipase by cGAMP leads to cell lysis, thereby limiting phage replication within the host population. Phage proteins that degrade cGAMP, sequester cGAMP, or prevent cGAMP synthesis have been identified in phages to evade CBASS protection. However, some phages are resistant to CBASS protection despite not encoding these proteins, suggesting that additional counter-defense mechanisms exist. To identify new anti-CBASS mechanisms in phages, we exploited bacterial host sensitivity to the antimicrobial compound sulfamethoxazole (SMX) as an indirect read-out for CBASS activity. Bacteria with active CBASS are more sensitive to SMX; therefore, we postulated that any viral proteins produced inside the bacterial host that target CBASS could increase bacterial SMX resistance. Using this screen, we identified that the Gp15 protein from the vibriophage ICP3 increases SMX resistance in Vibrio cholerae El Tor biotype that encodes an active CBASS. Gp15 is an essential virion protein involved in DNA ejection and conserved in many phages. Expression of Gp15 of another coliphage increases SMX resistance in Escherichia coli expressing an active CBASS, and it allows CBASS-sensitive phage to infect the bacterial host with CBASS, demonstrating an important role of Gp15 in phage defense evasion. Unlike other known anti-CBASS proteins, Gp15 does not degrade or sequester cGAMP, and it does not reduce the cellular level of cGAMP. Gp15 also does not interfere with CapV binding to cGAMP. Instead, Gp15 directly inhibits cGAMP-activated CapV serine hydrolase activity in a stoichiometric manner. Together, our study reveals a new mechanism for CBASS antagonism, in which a phage DNA ejection protein moonlights to inhibit a CBASS effector, thereby evading anti-phage defense.

microbiology↗

bioRxiv2025

Single-cell imaging reveals spontaneous phenotypic sorting and bet-hedging in developing biofilms

How phenotypic heterogeneity shapes biofilm architecture and development remains poorly understood. Motivated by this, we developed imaging tools to track intracellular levels of c-di-GMP, a key second messenger that controls the motile-to-sessile transition, in developing Vibrio cholerae biofilms at single-cell resolution. We show that c-di-GMP levels spontaneously bifurcate into a bimodal distribution that forms a spatially sorted pattern: High-c-di-GMP cells dominate the biofilm core while low-c-di-GMP cells localize to the periphery. Combining single-lineage tracing, mutant analysis, and agent-based modeling, we reveal that this pattern arises from differential viscosity and surface friction mediated by matrix-dependent interactions between cells and their microenvironments. We demonstrate that this heterogeneity and phenotypic sorting enable continuous emergence and shedding of planktonic cells, enhancing fitness in fluctuating environments. Our findings uncover a differential drag mechanism for pattern formation in multicellular systems, and expand the classical picture of biofilm lifecycle by highlighting the functional significance of phenotypic heterogeneity.

biophysics↗

bioRxiv2025

A horizontally acquired cyclic di-GMP phosphodiesterase regulated by zinc and quorum sensing

The second messenger cyclic di-GMP (cdG) in V. cholerae is indispensable for the regulation of biofilm formation, motility, and a variety of other important bacterial behaviors in the majority of bacteria. The human pathogen Vibrio cholerae has a diverse repertoire of diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes that control the intracellular cdG concentration depending on local environmental cues and its physiological state. Determining the transcriptional regulation of these enzymes and the respective environmental signals that control their activity is important to understand how and when V. cholerae switches between motile and sessile lifestyles in different environments. In some strains of the current V. cholerae 7th pandemic El Tor biotype, the horizontally acquired Vibrio Seventh Pandemic 2 (VSP-2) island encodes an uncharacterized PDE at the gene locus vc0515 which we named zpdA (Zinc-inhibited Phosphodiesterase-A). We show here that zpdA transcription is repressed by Zur when Zn2+ is abundant, as well as by the quorum sensing regulator HapR when cells grow to high density. Furthermore, we find that the PDE activity of the purified ZpdA protein is inhibited by Zn2+ but is dependent on alternative divalent cations such as Mn2+, which we find is elevated in V. cholerae cells grown under zinc limiting conditions. We conclude that ZpdA is an active metal-dependent PDE that is regulated by Zn2+ availability at both the level of transcription and post-translation leading to elevated cdG levels when Zn2+ is abundant. Our results demonstrate the important role of metal availability in modulating cdG signaling in bacteria. ImportanceV. cholerae colonizes estuarine environments and human host where it transitions between motile to sessile states which are controlled by cdG levels. cdG levels change in response to a variety of signals and are controlled by the activity of DGCs and PDEs, enzymes that make and degrade cdG. In this work, we show that Zn2+ and the cell density regulator HapR repress ZpdA, the PDE present in the VSP-2 island, at the level of transcription, and that Zn2+unexpectedly alters the PDE activity of ZpdA protein itself. Our study highlights the role of metal availability as an important signaling cue that controls V. cholerae biology.

microbiology↗

bioRxiv2025

DNA repair is essential for Vibrio cholerae growth on Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) Medium

Thiosulfate-citrate-bile salts-sucrose (TCBS) agar is a selective and differential media for the enrichment of pathogenic Vibrios. We observed that an exonuclease VII (exoVII) mutant of Vibrio cholerae failed to grow on TCBS agar, suggesting that DNA repair mutant strains may be hampered for growth in this selective media. Examination of the selective components of TCBS revealed that bile acids were primarily responsible for toxicity of the exoVII mutant. Suppressor mutations in DNA gyrase restored growth of the exoVII mutants on TCBS, suggesting that TCBS inhibits DNA gyrase similar to the antibiotic ciprofloxacin. To better understand what factors are important for V. cholerae to grow on TCBS, we generated a randomly-barcoded TnSeq (RB-TnSeq) library in V. cholerae and have used it to uncover a range of DNA repair mutants that also fail to grow on TCBS agar. The results of this study suggest that TCBS agar causes DNA damage to V. cholerae similarly to the mechanism of action of fluoroquinolones, and overcoming this DNA damage is critical for Vibrio growth on this selective medium. Abstract ImportanceTCBS is often used to diagnose cholera infection. We found that many mutant V. cholerae strains are attenuated for growth on TCBS agar, meaning they could remain undetected using this culture-dependent method. Hypermutator strains with defects in DNA repair pathways might be especially inhibited by TCBS. In addition, V. cholerae grown successively on TCBS agar develops resistance to ciprofloxacin.

microbiology↗

bioRxiv2024

Negative feedback of cyclic di-GMP levels optimizes switching between sessile and motile lifestyles in Vibrio cholerae

The signaling molecule cyclic di-GMP (c-di-GMP) controls the switch between bacterial motility and biofilm production, and fluctuations in cellular levels of c-di-GMP have been implicated in Vibrio cholerae pathogenesis. Intracellular concentrations of c-di-GMP are controlled by the interplay of diguanylate cyclase (DGC) enzymes, which synthesize c-di-GMP to promote biofilms, and phosphodiesterase (PDE) enzymes, which hydrolyze c-di-GMP to drive motility. To track the complete regulatory logic of how V. cholerae responds to changing c-di-GMP levels, we followed a time course of overexpression of either the V. campbellii diguanylate cyclase QrgB or a variant of QrgB lacking catalytic activity (QrgB*). We find that QrgB increases c-di-GMP levels relative to QrgB* for 30 minutes after overexpression, but the effect of QrgB on c-di-GMP levels plateaus at 30 minutes, indicating tight adaptive control of c-di-GMP levels. In contrast, loss of VpsR, a master regulator activating biofilm formation upon binding to c-di-GMP, leads to higher baseline levels of c-di-GMP and continuously increasing c-di-GMP through 60 minutes after QrgB induction, revealing the existence of a negative feedback loop on c-di-GMP levels operating through VpsR. Through a combination of RNA polymerase ChIP-seq, RNA-seq, and genetic approaches, we show that transcription of a gene encoding a PDE, cdgC, is activated by VpsR at high c-di-GMP concentrations, mediating this negative feedback on c-di-GMP levels. Further, although cells lacking cdgC exhibit enhanced biofilm formation, these mutants are outcompeted by wild type V. cholerae in colonization assays that reward a combination of attachment, dispersal, and motility behaviors. These results underscore the importance of negative feedback regulation of c-di-GMP to maintain appropriate homeostatic levels for efficient transitioning between biofilm formation and motility, both of which are necessary over the course of the V. cholerae infection cycle.

microbiology↗

bioRxiv2024

A Vibrio cholerae Type IV restriction system targets glucosylated 5-hydroxyl methyl cytosine to protect against phage infection

A major challenge faced by Vibrio cholerae is constant predation by bacteriophage (phage) in aquatic reservoirs and during infection of human hosts. To overcome phage predation, V. cholerae has evolved a myriad of phage defense systems. Although several novel defense systems have been discovered, we hypothesized more were encoded in V. cholerae given the relative paucity of phage that have been isolated which infect this species. Using a V. cholerae genomic library, we identified a Type IV restriction system consisting of two genes within a 16kB region of the Vibrio pathogenicity island-2 that we name TgvA and TgvB (Type I-embedded gmrSD-like system of VPI-2). We show that both TgvA and TgvB are required for defense against T2, T4, and T6 by targeting glucosylated 5-hydroxymethylcytosine (5hmC). T2 or T4 phages that lose the glucose modification are resistant to TgvAB defense but exhibit a significant evolutionary tradeoff becoming susceptible to other Type IV restriction systems that target unglucosylated 5hmC. We show that additional phage defense genes are encoded in VPI-2 that protect against other phage like T3, sec{Phi}18, sec{Phi}27 and {lambda}. Our study uncovers a novel Type IV restriction system in V. cholerae, increasing our understanding of the evolution and ecology of V. cholerae while highlighting the evolutionary interplay between restriction systems and phage genome modification. Abstract ImportanceBacteria are constantly being predated by bacteriophage (phage). To counteract this predation, bacteria have evolved a myriad of defense systems. Some of these systems specifically digest infecting phage by recognizing unique base modifications present on the phage DNA. Here, we discover a Type IV restriction system encoded in V. cholerae that we name TgvAB and demonstrate it recognizes and restricts phage that have 5-hydroxymethylcytosine glucosylated DNA. Moreover, the evolution of resistance to TgvAB render phage susceptible to other Type IV restriction systems, demonstrating a significant evolutionary tradeoff. These results enhance our understanding of the evolution of V. cholerae and more broadly how bacteria evade phage predation.

microbiology↗

bioRxiv2024

pGpG-signaling regulates virulence and global transcriptomic targets in Erwinia amylovora

Cyclic-di-GMP (c-di-GMP) is a critical bacterial second messenger that enables the physiological phase transition in Erwinia amylovora, the phytopathogenic bacterium that causes fire blight disease. C-di-GMP generation is dependent on diguanylate cyclase enzymes while the degradation of c-di-GMP can occur through the action of phosphodiesterase (PDE) enzymes that contain an active EAL and/or a HD-GYP domain. The HD-GYP-type PDEs, which are absent in E. amylovora, can directly degrade c-di-GMP into two GMP molecules. PDEs that contain an active EAL domain, as found in all active PDEs in E. amylovora, degrade c-di-GMP into pGpG. The signaling function of pGpG is not fully understood in bacterial systems. A transcriptomic approach revealed that elevated levels of pGpG in E. amylovora impacted several genes involved in metabolic and regulatory functions including several type III secretion and extracellular appendage related genes. The heterologous overexpression of an EAL or HD-GYP-type PDE in different background E. amylovora strains with varying c-di-GMP levels revealed that in contrast to the generation of pGpG, the direct breakdown of c-di-GMP into GMP by the HD-GYP-type PDE led to an elevation in amylovoran production and biofilm formation despite a decrease in c-di-GMP levels. The breakdown of c-di-GMP into pGpG (as opposed to GTP) also led to a decrease in virulence in apple shoots. The expression of hrpS was significantly increased in response to the breakdown of c-di-GMP into pGpG. Further, our model suggests that a balance in the intracellular ratio of pGpG and c-di-GMP is essential for biofilm regulation in E. amylovora. Importancec-di-GMP is the keystone molecule for regulating the transition from motility to biofilm formation in most bacteria. Interestingly, there are two distinct enzymatic phosphodiesterase (PDE) domains, termed EAL and HD-GYP, that degrade c-di-GMP. EAL domains cleave one bond of the cyclic ring to generate pGpG while HD-GYP enzymes cleave c-di-GMP to two GMP molecules. A central question regarding c-di-GMP signaling is has whether or not pGpG itself functions as a signaling molecule. Here we demonstrate in the plant pathogen Erwinia amylovora that pGpG specifically regulates genes and contributes to biofilm formation and disease progression.

microbiology↗

bioRxiv2023

Deployment of a Vibrio cholerae ordered transposon mutant library in a quorum-competent genetic background

Vibrio cholerae, the causative agent of cholera, has sparked seven pandemics in recent centuries, with the current one being the most prolonged. V. choleraes pathogenesis hinges on its ability to switch between low and high cell density gene regulatory states, enabling transmission between host and the environment. Previously, a transposon mutant library for V. cholerae was created to support investigations aimed toward uncovering the genetic determinants of its pathogenesis. However, subsequent sequencing uncovered a mutation in the gene luxO of the parent strain, rendering mutants unable to exhibit high cell density behaviors. In this study, we used chitin-independent natural transformation to move transposon insertions from these low cell density mutants into a wildtype genomic background. Library transfer was aided by a novel gDNA extraction we developed using thymol, which also showed high lysis-specificity for Vibrio. The resulting Grant Library comprises 3,102 unique transposon mutants, covering 79.8% of V. choleraes open reading frames. Whole genome sequencing of randomly selected mutants demonstrates 100% precision in transposon transfer to cognate genomic positions of the recipient strain. Notably, in no instance did the luxO mutation transfer into the wildtype background. Our research uncovered density-dependent epistasis in growth on inosine, an immunomodulatory metabolite secreted by gut bacteria that is implicated in enhancing gut barrier functions. Additionally, Grant Library mutants retain the plasmid that enables rapid, scarless genomic editing. In summary, the Grant Library reintroduces organismal relevant genetic contexts absent in the low cell density locked library equivalent. Significance StatementOrdered transposon mutant libraries are essential tools for catalyzing research by providing access to null mutants of all non-essential genes. Such a library was previously generated for Vibrio cholerae, but whole genome sequencing revealed that this library was made using a parent strain that is unable to exhibit cell-cell communication known as quorum sensing. Here, we utilize natural competence combined with a novel, high-throughput genomic DNA extraction method to regenerate the signaling incompetent V. cholerae ordered transposon mutant library in quorum sensing competent strain. Our library provides researchers with a powerful tool to understand V. cholerae biology within a genetic context that influences how it transitions from an environmentally benign organism to a disease-causing pathogen.

microbiology↗

bioRxiv2023

Targeting the Microbiome to Improve Gut Health and Breathing Function After Spinal Cord Injury

Spinal cord injury (SCI) is a devastating condition characterized by impaired motor and sensory function, as well as internal organ pathology and dysfunction. This internal organ dysfunction, particularly gastrointestinal (GI) complications, and neurogenic bowel, can reduce the quality of life of individuals with an SCI and potentially hinder their recovery. The gut microbiome impacts various central nervous system functions and has been linked to a number of health and disease states. An imbalance of the gut microbiome, i.e., gut dysbiosis, contributes to neurological disease and may influence recovery and repair processes after SCI. Here we examine the impact of high cervical SCI on the gut microbiome and find that transient gut dysbiosis with persistent gut pathology develops after SCI. Importantly, probiotic treatment improves gut health and respiratory motor function measured through whole-body plethysmography. Concurrent with these improvements was a systemic decrease in the cytokine tumor necrosis factor-alpha and an increase in neurite sprouting and regenerative potential of neurons. Collectively, these data reveal the gut microbiome as an important therapeutic target to improve visceral organ health and respiratory motor recovery after SCI. Research HighlightsO_LICervical spinal cord injury (SCI) causes transient gut dysbiosis and persistent gastrointestinal (GI) pathology. C_LIO_LITreatment with probiotics after SCI leads to a healthier GI tract and improved respiratory motor recovery. C_LIO_LIProbiotic treatment decreases systemic tumor necrosis factor-alpha and increases the potential for sprouting and regeneration of neurons after SCI. C_LIO_LIThe gut microbiome is a valid target to improve motor function and secondary visceral health after SCI. C_LI

neuroscience↗

bioRxiv2023

Plasmid-free cheater cells commonly evolve during laboratory growth

It has been nearly a century since the isolation and use of penicillin, heralding the discovery of a wide range of different antibiotics. In addition to clinical applications, such antibiotics have been essential laboratory tools, allowing for selection and maintenance of laboratory plasmids that encode cognate resistance genes. However, antibiotic resistance mechanisms can additionally function as public goods. For example, secretion of beta-lactamase from resistant cells, and subsequent degradation of nearby penicillin and related antibiotics, allows neighboring plasmid-free susceptible bacteria to survive antibiotic treatment. How such cooperative mechanisms impact selection of plasmids during experiments in laboratory conditions is poorly understood. Here, we show that the use of plasmid-encoded beta-lactamases leads to significant curing of plasmids in surface grown bacteria. Furthermore, such curing was also evident for aminoglycoside phosphotransferase and tetracycline antiporter resistance mechanisms. Alternatively, antibiotic selection in liquid growth led to more robust plasmid maintenance, although plasmid loss still occurred. The net outcome of such plasmid loss is the generation of a heterogenous population of plasmid-containing and plasmid-free cells, leading to experimental confounds that are not widely appreciated. ImportancePlasmids are routinely used in microbiology as readouts of cell biology or tools to manipulate cell function. Central to these studies is the assumption that all cells in an experiment contain the plasmid. Plasmid maintenance in a host cell typically depends on a plasmid-encoded antibiotic resistance marker, which provides a selective advantage when the plasmid containing cell is grown in the presence of antibiotic. Here we find that growth of plasmid-containing bacteria during laboratory conditions in the presence of three distinct antibiotic families leads to the evolution of a significant number of plasmid-free cells, which rely on the resistance mechanisms of the plasmid-containing cells for viability. This process generates a heterogenous population of plasmid-free and plasmid-containing bacteria, an outcome which could confound further experimentation.

microbiology↗

bioRxiv2023

Activation of a Vibrio cholerae CBASS anti-phage system by quorum sensing and folate depletion

A major challenge faced by bacteria is infection by bacteriophage (phage). Abortive infection is one strategy for combating phage in which an infected cell kills itself to limit phage replication, thus protecting neighboring kin. One class of abortive infection systems is the cyclic oligonucleotide based anti-phage signaling system (CBASS) which relies on two core enzymatic activities; an oligo-nucleotide cyclase that is activated following phage infection and a cyclic-oligo-nucleotide sensitive effector whose activity kills the infected cell. However, the mechanisms behind the deployment and activation of these lethal CBASS systems prior-to and following infection have largely remained a mystery. While exploring unique genomic features of the current pandemic Vibrio cholerae biotype El Tor for clues underlying its pandemic success we found its CBASS was spuriously activated by the folate biosynthesis inhibitor sulfamethoxazole, but only after the population had reached a high-cell density. This population density dependent activity revealed that transcription of both the oligo-nucleotide cyclase, dncV, and the CBASS phospholipase effector, capV, is enhanced at high-cell density by quorum sensing. Together, these results demonstrate that the V. cholerae CBASS is deployed when the environment is densely populated and activated in response to a perturbation in folate biosynthesis. SignificanceTo counteract infection with phage, bacteria have evolved a myriad of molecular defense systems. Some of these systems initiate a process called abortive infection, in which the infected cell kills itself to prevent phage propagation. However, such systems must be inhibited in the absence of phage infection to prevent spurious death of the host. Here we show that the cyclic oligonucleotide based anti-phage signaling system (CBASS) accomplishes this by sensing intracellular folate molecules and only expressing this system in a group. These results enhance our understanding of the evolution of the 7th V. cholerae pandemic and more broadly how bacteria defend themselves against phage infection.

microbiology↗

bioRxiv2023

Time to lysis determines phage sensitivity to a cytidine deaminase toxin/antitoxin bacterial defense system

Toxin-antitoxin (TA) systems are ubiquitous two-gene loci that bacteria use to regulate cellular processes such as phage defense. Here, we demonstrate the mechanism by which a novel type III TA system, avcID, is activated and confers resistance to phage infection. The toxin of the system (AvcD) is a deoxycytidylate deaminase that converts deoxycytidines (dC) to dexoyuridines (dU), while the RNA antitoxin (AvcI) inhibits AvcD activity. We have shown that AvcD deaminated dC nucleotides upon phage infection, but the molecular mechanism that activated AvcD was unknown. Here we show that the activation of AvcD arises from phage-induced shutoff of host transcription, leading to degradation of the labile AvcI. AvcD activation and nucleotide depletion not only decreases phage replication but also increases the formation of defective phage virions. Surprisingly, infection of phages such as T7 that are not inhibited by AvcID also lead to AvcI RNA antitoxin degradation and AvcD activation, suggesting that depletion of AvcI is not sufficient to confer protection against some phage. Rather, our results support that phage with a longer lysis time like T5 are sensitive to AvcID-mediated protection while those with a shorter lysis time like T7 are resistant. AUTHORS SUMMARYNumerous diverse antiphage defense systems have been discovered in the past several years, but the mechanisms of how these systems are activated upon phage infection and why these systems protect against some phage but not others are poorly understood. The AvcID toxin-antitoxin phage defense system depletes nucleotides of the dC pool inside the host upon phage infection. We show that phage inhibition of host cell transcription activates this system by depleting the AvcI inhibitory sRNA, which inhibits production of phage and leads to the formation of defective virions. Additionally, we determined that phage lysis time is a key factor that influences sensitivity to AvcID with faster replicating phage exhibiting resistance to its effects. This study has implications for understanding the factors that influence bacterial host/phage dynamics.

microbiology↗

bioRxiv2023

Three dominant Vibrio cholerae lytic phage all require O1 antigen for infection

In its natural aquatic environment, the bacterial pathogen Vibrio cholerae, the causative agent of the enteric disease cholera, is in constant competition with bacterial viruses known as phages. Following ICP3 infection, V. cholerae cultures that exhibited phage killing always recovered overnight, and clones isolated from these regrowth populations exhibited complete resistance to subsequent infections. Whole genome sequencing of these resistant mutants revealed seven distinct mutations in genes encoding for enzymes involved in O1 antigen biosynthesis, demonstrating that the O1 antigen is a previously uncharacterized putative receptor of ICP3. To further elucidate the specificity of the resistance conferred by these mutations, they were challenged with the V. cholerae-specific phages ICP1 and ICP2. All seven O1 antigen mutants demonstrated pan-resistance to ICP1 but not ICP2, which utilizes the OmpU outer membrane protein as a receptor. We show that resistant mutations to ICP1 and ICP3 evolve at a significantly higher frequency than ICP2, but these mutations have a significant fitness tradeoff to V. cholerae and are unable to evolve in the presence of an antimicrobial that mimics host cell defensins.

microbiology↗

bioRxiv2021

SARS-CoV-2 Spike Protein Regulation of Angiotensin Converting Enzyme 2 and Tissue Renin-Angiotensin Systems: Influence of Biologic Sex

Angiotensin converting enzyme 2 (ACE2) is an enzyme that limits activity of the renin-angiotensin system (RAS) and also serves as a receptor for the SARS-CoV-2 Spike (S) protein. Binding of S protein to ACE2 causes internalization which activates local RAS. ACE2 is on the X chromosome and its expression is regulated by sex hormones. In this study, we defined ACE2 mRNA abundance and examined effects of S protein on ACE2 activity and/or angiotensin II (AngII) levels in pivotal tissues (lung, adipose) from male and female mice. In lung, ACE2 mRNA abundance was reduced following gonadectomy (GDX) of male and female mice and was higher in XX than XY mice of the Four Core Genotypes (FCG). Reductions in lung ACE2 mRNA abundance by GDX occurred in XX, but not XY FCG female mice. Lung mRNA abundance of ADAM17 and TMPRSS2, enzymes that shed cell surface ACE2 and facilitate viral cell entry, was reduced by GDX in male but not female mice. For comparison, adipose ACE2 mRNA abundance was higher in female than male mice and higher in XX than XY FCG mice. Adipose ADAM17 mRNA abundance was increased by GDX of male and female mice. S protein reduced ACE2 activity in alveolar type II epithelial cells and 3T3-L1 adipocytes. Administration of S protein to male and female mice increased lung AngII levels and decreased adipose ACE2 activity in male but not female mice. These results demonstrate that sex differences in ACE2 expression levels may impact local RAS following S protein exposures.

physiology↗

bioRxiv2021

Cervical Spinal Cord Injury Leads to Injury and Metabolic Dysfunction of the Lung

High-cervical spinal cord injury (SCI) often disrupts respiratory motor pathways and disables breathing in the affected population. Moreover, cervically injured individuals are at risk for developing acute lung injury (ALI), which predicts substantial mortality rates. While the correlation between ALI and SCI has been found in the clinical setting, the field lacks an animal model to interrogate the fundamental biology of this relationship. To begin to address this gap, we performed an experimental cervical SCI and assessed lung injury in adult rats. We demonstrate that animals display signs of ALI two weeks post-SCI. We also observed aberrant N-glycan metabolism determined by matrix-assisted laser desorption/ionization mass spectrometry imaging. Collectively, we establish for the first time a model of ALI after SCI at an acute time point that can be used to monitor the progression of lung damage, as well as identify potential targets to ameliorate ALI.

neuroscience↗

bioRxiv2021

Multi-Dimensional Reduction Clustering of Complex Carbohydrates Reveal Tissue Metabolism, Heterogeneity and Histopathology

Formalin-fixed paraffin-embedded (FFPE) human tissues represent the world's largest collection of accessible clinical specimens with matched, well-annotated clinical course for disease progression. Currently, FFPE sections are limited to low throughput histo- and immunological assessments. Extracting largescale molecular information remains a major technological barrier to uncover the vast potential within FFPE specimens for translation and clinical research. Two critical but understudied facets of glucose metabolism are anabolic pathways for glycogen and N-linked glycan biosynthesis. Together, these complex carbohydrates represent bioenergetics, protein-structure function, and tissue architecture in human biology. Herein, we report the high-dimensional Metabolomics-Assisted Digital pathology Imaging (Madi) workflow that combines matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) with machine learning for the comprehensive assessment of tissue heterogeneity, histopathology, and metabolism in human FFPE sections. In normal human tissue sections, Madi accurately identifies anatomical regions within liver and the brain. In human lung diseases, Madi accurately predicts major lung pathologies such as honeycomb change, late-stage fibrosis, diffuse alveolar damage (DAD), and acute fibrinous and organizing pneumonia (AFOP) from idiopathic pulmonary fibrosis (IPF) and COVID-19 pneumonia specimens with precision. In depth pathway enrichment analyses reveal unique metabolic pathways are associated with distinct pathological regions, which highlight aberrant complex carbohydrate metabolism as a previously unknown molecular event associated with disease progression that could hold key to future therapeutic interventions.

systems biology↗