Biology subjects

Evans, D. J.

Publications and source records attributed to Evans, D. J..

12 recordsLinked to original sources

bioRxiv2025

Prior contact lens wear reduces susceptibility of the superficially-injured cornea to bacterial adhesion

Contact lens wear in humans and mice is consistently associated with asymptomatic corneal parainflammation. Here, we tested the hypothesis that the corneal response to lens wear alone can function to protect it against bacterial adhesion enabled by superficial injury. One eye only of mT/mG-LysMcre mice (cell membranes red; Lyz2+ cells green) wore a contact lens for 4-6 days. After lens removal, mice were anesthetized and both corneas superficially-injured before bacterial inoculation with either a mouse eyelid commensal (Macrococcus epidermidis) or a corneal pathogen (Pseudomonas aeruginosa). Inoculation was repeated hourly for 4 hours under anesthesia before euthanasia. Enucleated eyes were fixed overnight, and adherent bacteria visualized using a universal 16S rRNA-targeted FISH probe (M. epidermidis) or Blue Fluorescent Protein (P. aeruginosa). Confocal imaging and Imaris software were used to quantify bacterial adhesion and location in the epithelium, and also the number, location and morphology of Lyz2+ cells. For both commensal and P. aeruginosa, prior lens wear resulted in reduced adhesion to the superficially injured corneas ([~] 55% and [~] 45% respectively). In both instances this correlated with increased numbers of corneal Lyz2+ cells. Other details differed for the two bacterial types. For the commensal, prior lens wear resulted in bacteria penetrating deeper into the epithelium versus contralateral eyes, with Lyz2+ cells extending their processes further into the epithelium and localizing closer to the cornea surface. For P. aeruginosa, prior lens wear resulted in adherent bacteria closer to the cornea surface, and Lyz2+ cells moving further away from it. Moreover, while overall Lyz2+ cell sphericity increased for P. aeruginosa with prior lens wear versus contralateral eyes, it showed no overall change for the commensal. Lyz2+ cell volume in the central cornea was decreased for P. aeruginosa with prior lens wear but increased for the commensal. Thus, corneal responses to prior lens wear can quantitively reduce bacterial adhesion to superficially-injured corneas correlating with a Lyz2+ cell response for both a commensal and a pathogen, with differences in details for the two bacterial types. How continued lens wear supersedes this protective response to promote P. aeruginosa infection pathogenesis remains to be determined, as does the relationship to the Lyz2+ cell response. Author summaryContact lens wear induces asymptomatic corneal parainflammation in humans and mice involving multiple immune cell types. Here, we used a mouse model to test if previous lens wear would subsequently protect superficially-injured corneas against bacterial adhesion by either a murine commensal (Macrococcus epidermidis) or an opportunistic pathogen (Pseudomonas aeruginosa). Confocal imaging was used to quantify bacterial adhesion/location in addition to Lyz2+ immune cell numbers, location and morphology. Prior lens wear for 4-6 days, known to trigger corneal parainflammation, reduced the propensity for bacteria to adhere to corneas that were superficially-injured after the lens was removed: [~] 55% for the commensal and [~] 45% for P. aeruginosa versus contralateral eyes that had not worn a lens. In both instances this correlated with increased Lyz2+ immune cell numbers. However, differences were also noted, including that adherent commensals were found penetrating further into the epithelium while adherent P. aeruginosa remained close to the corneal surface. Lyz2+ cell location, sphericity and volume also differed for the two bacterial types. In sum, lens wear in the absence of bacterial inoculation can enable a response that protects the cornea against bacterial challenge after lens removal. How lens wear during inoculation overcomes this protective response to promote P. aeruginosa infection pathogenesis remains to be determined.

microbiology↗

bioRxiv2025

TRPV1 Defends the Healthy Murine Cornea against Staphylococcus aureus Adhesion Independently of Sensory Nerve Firing

PurposePreviously we showed that transient receptor potential ion channels TRPA1 and TRPV1 selectively protect the cornea against bacterial adhesion, with TRPA1 countering the Gram-negative pathogen Pseudomonas aeruginosa and TRPV1 countering environmental bacteria. Here, we explored parameters of this specificity using a Gram-positive pathogen Staphylococcus aureus. MethodsHealthy corneas of C57BL/6J wild-type (WT), TRPA1 (-/-) or TRPV1 (-/-) mice were challenged with S. aureus for 4 or 6 hours. Some experiments instead/also used resiniferatoxin (RTX) to deplete TRPV1-expressing nerves, JNJ-17203212 to selectively antagonize TRPV1, or the anesthetic bupivacaine to inhibit nerve firing. Adherent bacteria were quantified using FISH labeling (16S rRNA-targeted probe). Lyz2+, CD11c+ and CD45+ cells were visualized/quantified using hybrid mT/mG + LysMcre mice (red cell membranes; Lyz2+-GFP), CD11c+-YFP mice, and anti-CD45-antibody respectively. ResultsCorneas of TRPV1 (-/-) not TRPA1 (-/-) mice were found more susceptible to S. aureus adhesion compared to WT. Accordingly, either ablation of TRPV1-expressing nerves or TRPV1 antagonism increased adhesion. Defense against S. aureus adhesion did not depend on nerve firing. Despite having no significant impact on CD11c+ or Lyz2+ cell numbers, S. aureus challenge increased CD45+ cell counts, also dependent on TRPV1-expressing nerves, and it increased Lyz2+ cell sphericity and volume. ConclusionHealthy corneas utilize TRPV1 to protect against S. aureus adhesion independently of sensory nerve firing. This contrasts with defense against P. aeruginosa adhesion which requires TRPA1 and nerve firing. How the differential immune cell responses to these two pathogens relate to TRP-dependent defense against adhesion remains to be determined.

microbiology↗

bioRxiv2025

Cross-membrane cooperation among bacteria can facilitate intracellular pathogenesis

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen able to cause life- and sight-threating infections. Once considered an extracellular pathogen, numerous studies have shown it can survive intracellularly. Previously, we showed that P. aeruginosa inside cells can diversify into distinct subpopulations in vacuoles and the cytoplasm. Here, we report that the transition from vacuoles to cytoplasm requires collaboration with the extracellular subpopulation, through Ca2+ influx enabled by their type III secretion system (T3SS) translocon pore proteins. Moreover, we show that collaboration among P. aeruginosa subpopulations can contribute to disseminating intracellular bacteria in vivo in a mouse infection model. This study provides the basis for future studies to investigate how cooperation of extracellular and intracellular bacteria within the host may contribute to disease progression and persistence.

microbiology↗

bioRxiv2025

The Pseudomonas aeruginosa T3SS can contribute to traversal of an in situ epithelial multilayer independently of the T3SS needle

Multilayered epithelia lining our tissue surfaces normally resist traversal by opportunistic bacteria. Previously, we developed a strategy to experimentally perturbate this resistance in situ in the corneas of mouse eyes and used it to show that traversal of a multilayered epithelium by Pseudomonas aeruginosa requires ExsA, the transcriptional activator of its type 3 secretion system (T3SS). Here, we developed a novel strategy for quantitively localizing individual traversing bacteria within the in situ multilayered corneal epithelium and explored contributions of T3SS components. The results showed that T3SS translocon and T3SS effector mutants had reduced epithelial traversal efficiency. Surprisingly, a {Delta}pscC mutant unable to assemble the T3SS needle traversed as efficiently as wild-type P. aeruginosa, while a {Delta}exsD mutant constitutively on for T3SS expression was traversal defective. Dispensability of the T3SS needle for effector-mediated traversal was confirmed using a mutant lacking the T3SS operon except the effector genes ({Delta}pscU-L mutant). That mutant reacquired the ability to traverse if complemented with rhamnose-inducible exsA, but not if the effector genes were also deleted ({Delta}pscU-L{Delta}exoSTY). Western immunoblot confirmed ExoS in culture supernatants of rhamnose-induced exsA-complemented {Delta}pscU-L mutants lacking all T3SS needle protein genes. Together, these results show that epithelial traversal by P. aeruginosa can involve T3SS effectors and translocon proteins independently of the T3SS needle previously thought essential for T3SS function. This advances our understanding of P. aeruginosa pathogenesis and has relevance to development of therapeutics targeting the T3SS system. IO_SCPLOWMPORTANCEC_SCPLOWWhile the capacity to cross an epithelial barrier can be a critical step in bacterial pathogenesis, our understanding of mechanisms involved is derived largely from cell culture experimentation. The latter is due to practical limitations of in vivo/in situ models and challenge of visualizing individual bacteria in the context of host tissue. Here, factors used by P. aeruginosa to traverse an epithelial multilayer in situ were studied by: 1) leveraging the transparent properties and superficial location of the cornea, 2) using our established method for enabling bacterial traversal susceptibility, and 3) developing a novel strategy for accurate and quantitative localization of individual traversing bacteria in situ. Outcomes showed that T3SS translocon and T3SS effector proteins synergistically contribute to epithelial traversal efficiency independently of the T3SS needle. These findings challenge the assumption that the T3SS needle is essential for T3SS effectors or translocon proteins to contribute to bacterial pathogenesis.

microbiology↗

bioRxiv2024

Contact Lens Wear Alters Transcriptional Responses to Pseudomonas aeruginosa in Both the Corneal Epithelium and the Bacteria

PurposeHealthy corneas resist colonization by virtually all microbes yet contact lens wear can predispose the cornea to sight-threatening infection with Pseudomonas aeruginosa. Here, we explored how lens wear changes corneal epithelium transcriptional responses to P. aeruginosa and its impact on bacterial gene expression. MethodsMale and female C57BL/6J mice were fitted with a contact lens on one eye for 24 h. After lens removal, corneas were immediately challenged for 4 h with P. aeruginosa. A separate group of naive mice were similarly challenged with bacteria. Bacteria-challenged eyes were compared to uninoculated naive controls as was lens wear alone. Total RNA-sequencing determined corneal epithelium and bacterial gene expression. ResultsPrior lens wear profoundly altered the corneal response to P. aeruginosa, including: upregulated pattern-recognition receptors (tlr3, nod1), downregulated lectin pathway of complement activation (masp1), amplified upregulation of tcf7, gpr55, ifi205, wfdc2 (immune defense) and further suppression of efemp1 (corneal stromal integrity). Without lens wear, P. aeruginosa upregulated mitochondrial and ubiquinone metabolism genes. Lens wear alone upregulated axl, grn, tcf7, gpr55 (immune defense) and downregulated Ca2+-dependent genes necab1, snx31 and npr3. P. aeruginosa exposure to prior lens wearing vs. naive corneas upregulated bacterial genes of virulence (popD), its regulation (rsmY, PA1226) and antimicrobial resistance (arnB, oprR). ConclusionPrior lens wear impacts corneal epithelium gene expression altering its responses to P. aeruginosa and how P. aeruginosa responds to it favoring virulence, survival and adaptation. Impacted genes and associated networks provide avenues for research to better understand infection pathogenesis.

microbiology↗

bioRxiv2024

SARS-CoV-2 3CLPro Dihedral Angles Reveal Allosteric Signaling

In allosteric proteins, identifying the pathways that signals take from allosteric ligand-binding sites to enzyme active sites or binding pockets and interfaces remains challenging. This avenue of research is motivated by the goals of understanding particular macromolecular systems of interest and creating general methods for their study. An especially important protein that is the subject of many investigations in allostery is the SARS-CoV-2 main protease (Mpro), which is necessary for coronaviral replication. It is both an attractive drug target and, due to intense interest in it for the development of pharmaceutical compounds, a gauge of the state-of-the-art approaches in studying protein inhibition. Here we develop a computational method for characterizing protein allostery and use it to study Mpro. We propose a role of the proteins C-terminal tail in allosteric modulation and warn of unintuitive traps that can plague studies of the role of protein dihedrals angles in transmitting allosteric signals.

biophysics↗

bioRxiv2023

Intracellular replication of Pseudomonas aeruginosa in epithelial cells requires suppression of the caspase-4 inflammasome

Pathogenesis of Pseudomonas aeruginosa infections can include bacterial survival inside epithelial cells. Previously, we showed this involves multiple roles played by the type three-secretion system (T3SS), and specifically the effector ExoS. This includes ExoS-dependent inhibition of a lytic host cell response that subsequently enables intracellular replication. Here, we studied the underlying cell death response to intracellular P. aeruginosa, comparing wild-type to T3SS mutants varying in capacity to induce cell death and that localize to different intracellular compartments. Results showed that corneal epithelial cell death induced by intracellular P. aeruginosa lacking the T3SS, which remains in vacuoles, correlated with activation of NF-{kappa}B as measured by p65 relocalization and TNF transcription and secretion. Deletion of caspase-4 through CRISPR-Cas9 mutagenesis delayed cell death caused by these intracellular T3SS mutants. Caspase-4 deletion also countered more rapid cell death caused by T3SS effector-null mutants still expressing the TSSS apparatus that traffic to the host cell cytoplasm, and in doing so rescued intracellular replication normally dependent on ExoS. While HeLa cells lacked a lytic death response to T3SS mutants, it was found to be enabled by interferon gamma treatment. Together, these results show that epithelial cells can activate the noncanonical inflammasome pathway to limit proliferation of intracellular P. aeruginosa, not fully dependent on bacterially-driven vacuole escape. Since ExoS inhibits the lytic response, the data implicate targeting of caspase-4, an intracellular pattern recognition receptor, as another contributor to the role of ExoS in the intracellular lifestyle of P. aeruginosa. ImportancePseudomonas aeruginosa can exhibit an intracellular lifestyle within epithelial cells in vivo and in vitro. The type three secretion system (T3SS) effector ExoS contributes via multiple mechanisms, including extending the life of invaded host cells. Here, we aimed to understand the underlying cell death inhibited by ExoS when P. aeruginosa is intracellular. Results showed that intracellular P. aeruginosa lacking T3SS effectors could elicit rapid cell lysis via the non-canonical inflammasome pathway. Caspase-4 contributed to cell lysis even when the intracellular bacteria lacked the entire T33S and were consequently unable to escape vacuoles, representing a naturally occurring subpopulation during wildtype infection. Together, the data show the caspase-4 inflammasome as an epithelial cell defense against intracellular P. aeruginosa, and implicate its targeting as another mechanism by which ExoS preserves the host cell replicative niche.

microbiology↗

bioRxiv2022

Pseudomonas aeruginosa can diversify after host cell invasion to establish multiple intracellular niches

Within epithelial cells, Pseudomonas aeruginosa depends on its type three secretion system (T3SS) to escape vacuoles and replicate rapidly in the cytosol. Previously, it was assumed that intracellular subpopulations remaining T3SS-negative (and therefore in vacuoles) were destined for degradation in lysosomes, supported by data showing vacuole acidification. Here, we report in both corneal and bronchial human epithelial cells that vacuole associated-bacteria can persist, sometimes in the same cells as cytosolic bacteria. Using a combination of phase-contrast, confocal, and correlative light and electron microscopy, we also found they can demonstrate biofilm-associated markers: cdrA and cyclic-di-GMP (c-di-GMP). Vacuolar-associated bacteria, but not cytosolic counterparts, tolerated the cell-permeable antibiotic ofloxacin. Surprisingly, use of mutants showed that both persistence in vacuoles and ofloxacin tolerance were independent of the biofilm-associated protein CdrA or exopolysaccharides (Psl, Pel, alginate). A T3SS mutant ({Delta}exsA) unable to escape vacuoles phenocopied vacuolar-associated sub-populations in wild-type PAO1-infected cells, results revealing that epithelial cell death depended upon bacterial viability. Intra-vital confocal imaging of infected mouse corneas confirmed that P. aeruginosa formed similar intracellular sub-populations within epithelial cells in vivo. Together, these results show that P. aeruginosa differs from other pathogens by diversifying intracellularly into vacuolar and cytosolic sub-populations that both contribute to pathogenesis. Their different gene expression and behavior (e.g., rapid replication versus slow replication/persistence) suggest cooperation favoring both short- and long-term interests and another potential pathway to treatment failure. How this intracellular diversification relates to previously described "acute versus chronic" virulence gene-expression phenotypes of P. aeruginosa remains to be determined. ImportancePseudomonas aeruginosa can cause sight- and life-threatening opportunistic infections, and its evolving antibiotic resistance is a growing concern. Most P. aeruginosa strains can invade host cells, presenting a challenge to therapies that do not penetrate host cell membranes. Previously, we showed that the P. aeruginosa type III secretion system (T3SS) plays a pivotal role in survival within epithelial cells, allowing escape from vacuoles, rapid replication in the cytoplasm, and suppression of host cell death. Here, we report the discovery of a novel T3SS-negative sub-population of intracellular P. aeruginosa within epithelial cells that persist in vacuoles rather than the cytoplasm, and that tolerate a cell-permeable antibiotic (ofloxacin) that is able to kill cytosolic bacteria. Classical biofilm-associated markers, although demonstrated by this sub-population, are not required for vacuolar persistence or antibiotic tolerance. These findings advance our understanding of how P. aeruginosa hijacks host cells, showing it diversifies into multiple populations with T3SS-negative members enabling persistence whilst rapid replication is accomplished by more vulnerable T3SS-positive siblings. Intracellular P. aeruginosa persisting and tolerating antibiotics independently of the T3SS or biofilm-associated factors could present additional challenges to development of more effective therapeutics.

microbiology↗

bioRxiv2021

Imprecise recombinant viruses evolve via a fitness-driven, iterative process of polymerase template-switching events.

Recombination is a common feature of many positive-strand RNA viruses, playing an important role in virus evolution. However, to date, there is limited understanding of the mechanisms behind the process. Utilising in vitro assays, we have previously shown that the template-switching event of recombination is a random and ubiquitous process that often leads to recombinant viruses with imprecise genomes containing sequence duplications. Subsequently, a process termed resolution, that has yet to be mechanistically studied, removes these duplicated sequences resulting in a virus population of wild type length genomes. Using defined imprecise recombinant viruses together with Oxford Nanopore and Illumina high throughput next generation sequencing technologies we have investigated the process of resolution. We show that genome resolution involves subsequent rounds of template-switching recombination with viral fitness resulting in the survival of a small subset of recombinant genomes. This alters our previously held understanding that recombination and resolution are independent steps of the process, and instead demonstrates that viruses undergo frequent and continuous recombination events over a prolonged period until the fittest viruses, predominantly those with wild type length genomes, dominate the population. Author SummaryViruses with positive-sense RNA genomes, such as poliovirus, have several mechanisms by which they evolve. One of these is the process of recombination involving the large-scale exchange of genetic information. Recombination occurs during replication when the viral polymerase switches from copying one genome to another. However, the polymerase does not always accurately switch between the two, resulting in sequence duplications or deletions, and genomes that are referred to as imprecise. Over multiple rounds of replication sequence duplications are lost and genomes are resolved to wild type length, but it is unclear how this occurs. Here we used synthetic polioviruses containing defined sequence duplications to determine that the genome population undergoes repeated rounds of recombination until sequence duplications are lost and viruses with precise, wild type length genomes are selected for. This selection is based on the overall fitness of the virus population, with less fit imprecise viruses evolving more quickly. Our study suggests that recombination is a continual process where virus fitness drives the selection of a small subset of recombinant variants. These data are important for understanding how novel viruses evolve via recombination and how this process can be blocked to prevent novel and dangerous pathogens from arising.

microbiology↗

bioRxiv2021

First come, first served: Superinfection exclusion in Deformed wing virus is dependent upon sequence identity and not the order of virus acquisition.

Deformed wing virus (DWV) is the most important globally distributed pathogen of honey bees and, when vectored by the ectoparasite Varroa destructor, is associated with high levels of colony losses. Divergent DWV types may differ in their pathogenicity and are reported to exhibit superinfection exclusion upon sequential infections, an inevitability in a Varroa-infested colony. We used a reverse genetic approach to investigate competition and interactions between genetically distinct or related virus strains, analysing viral load over time, tissue distribution with reporter gene-expressing viruses and recombination between virus variants. Transient competition occurred irrespective of the order of virus acquisition, indicating no directionality or dominance. Over longer periods, the ability to compete with a pre-existing infection correlated with the genetic divergence of the inoculae. Genetic recombination was observed throughout the DWV genome with recombinants accounting for ~2% of the population as determined by deep sequencing. We propose that superinfection exclusion, if it occurs at all, is a consequence of a cross-reactive RNAi response to the viruses involved, explaining the lack of dominance of one virus type over another. A better understanding of the consequences of dual- and superinfection will inform development of cross-protective honey bee vaccines and landscape-scale DWV transmission and evolution.

microbiology↗

bioRxiv2021

Finding Druggable Sites in Proteins using TACTICS

Structure-based drug discovery efforts require knowledge of where drug-binding sites are located on target proteins. To address the challenge of finding druggable sites, we developed a machine-learning algorithm called TACTICS (Trajectory-based Analysis of Conformations To Identify Cryptic Sites), which uses an ensemble of molecular structures (such as molecular dynamics simulation data) as input. First, TACTICS uses k-means clustering to select a small number of conformations that represent the overall conformational heterogeneity of the data. Then, TACTICS uses a random forest model to identify potentially bindable residues in each selected conformation, based on protein motion and geometry. Lastly, residues in possible binding pockets are scored using fragment docking. As proof-of-principle, TACTICS was applied to the analysis of simulations of the SARS-CoV-2 main protease and methyltransferase and the Yersinia pestis aryl carrier protein. Our approach recapitulates known small-molecule binding sites and predicts the locations of sites not previously observed in experimentally determined structures. The TACTICS code is available at https://github.com/Albert-Lau-Lab/tactics_protein_analysis.

biophysics↗

bioRxiv2019

Deformed Wing Virus spillover from honey bees to bumble bees: a reverse genetic study.

Deformed wing virus (DWV) is a persistent pathogen of European honey bees and the major contributor to overwintering colony losses. The prevalence of DWV in honey bees has led to significant concerns about spillover of the virus to other pollinating species. Bumble bees are both a major group of wild and commercially-reared pollinators. Several studies have reported pathogen spillover of DWV from honey bees to bumble bees, but evidence of a true sustained viral infection has yet to be demonstrated. Here we investigate the infectivity and transmission of DWV in bumble bees using the buff-tailed bumble bee Bombus terrestris as a model. We apply a reverse genetics approach combined with controlled laboratory conditions to detect and monitor DWV infection. A novel reverse genetics system for three representative DWV variants, including the two master variants of DWV - type A and B - was used. Our results directly confirm DWV replication in bumble bees but also demonstrate striking resistance to infection by certain routes. Bumble bees may support DWV replication but it is not clear how infection could occur under natural environmental conditions.

microbiology↗