Biology subjects

Bostina, M.

Publications and source records attributed to Bostina, M..

5 recordsLinked to original sources

bioRxiv2025

Enteropathogenic Escherichia coli manipulates the host exocyst complex to enhance pedestal formation

Enteropathogenic Escherichia coli (EPEC), a major cause of diarrheal disease, produces plasma membrane pedestals that promote colonization of host cells. Critical for pedestal formation is EPECs type III secretion system, which injects [~] 20 effector proteins into human cells. One of these effectors is Tir, which inserts into the host plasma membrane and stimulates the assembly of actin filaments essential for pedestal generation. To date, actin polymerization is the only host process known to contribute to pedestal formation. Here we report that EPEC co-opts the membrane trafficking pathway of polarized exocytosis, which acts together with actin polymerization to allow the efficient production of pedestals. Polarized exocytosis is mediated by the exocyst -- a human octameric complex that uses intracellular vesicles to expand the plasma membrane. We found that EPEC stimulated exocytosis at sites of pedestal formation in a manner dependent on the exocyst. The bacterial effector EspH recruited the exocyst and promoted exocytosis. Genetic inactivation of espH or RNA interference (RNAi)-induced depletion of exocyst components reduced both the frequency and size of pedestals. Additional RNAi experiments indicated that the exocyst is dispensable for actin filament assembly in pedestals. Co-depletion of components of the exocyst and the Arp2/3 complex showed that exocytosis and actin polymerization make additive contributions to pedestal formation. Collectively, these results indicate that exocyst-mediated expansion of the plasma membrane acts together with actin polymerization to optimize the generation of pedestals. IMPORTANCEEnteropathogenic E. coli (EPEC) induces the formation of host cell plasma membrane protrusions called "pedestals" that mediate tight adherence to intestinal epithelial cells. Substantial evidence indicates that the generation of pedestals requires bacterial-induced polymerization of the host cell actin cytoskeleton. However, it remains unknown if EPEC manipulates other human processes to augment the efficiency of pedestal generation. The significance of this research is to demonstrate that EPEC co-opts a second host process called polarized exocytosis to increase the frequency of pedestals and the size of these structures. EPEC induces exocytosis by using its effector protein EspH to recruit the human exocyst complex, which mediates the insertion of membrane at sites of bacterial attachment. Our results demonstrate EPECs ability to coordinate host membrane trafficking with cytoskeletal changes to enhance infection. Remarkably, EPEC shares this strategy with the intracellular bacterial pathogens Listeria monocytogenes and Shigella flexneri.

microbiology↗

bioRxiv2024

Journey to the center of the phage; revealing the ejectosome of Pectobacterium bacteriophage {Phi}M1

Podophages that infect gram-negative bacteria, such as Pectobacterium pathogen {Phi}M1, encode tail assemblies too short to extend across the complex gram-negative cell wall. To overcome this, podophages encode a large protein complex (ejectosome) packaged inside the viral capsid and correspondingly ejected during infection to form a transient channel that spans the periplasmic space. Here we describe the ejectosome of bacteriophage {Phi}M1 to a resolution of 3.32 [A] by single particle cryo-EM. The core consists of tetrameric and octameric ejection proteins which form a [~]1.5 MDa ejectosome that must transition through the [~]30 [A] aperture created by the short tail nozzle assembly that acts as the conduit for the passage of DNA during infection. The ejectosome forms several grooves into which coils of genomic DNA are fit before the DNA sharply turns and goes down the tunnel and into the portal. In addition, we reconstructed the icosahedral capsid and hybrid tail apparatus to resolutions between 3.04 [A] and 3.23 [A], and note an uncommon fold adopted by the dimerized decoration proteins which further emphasize the structural diversity of podophages. These reconstructions have allowed the generation of a complete atomic model of the {Phi}M1, uncovering two distinct decoration proteins and highlighting the exquisite structural diversity of tailed bacteriophages. Significance StatementThis study resolves the cryo-EM structure of bacteriophage {Phi}M1, which possesses several unique and interesting structural elements, including a pair of distinct decoration proteins that are underreported in tailed DNA phages. Significantly, we also report the internal ejectosome proteins of {Phi}M1, which are highly non-conserved with previously solved proteins to date and demonstrate the structural diversity of ejection proteins. The ejectosome reveals a DNA spooling phenomenon whereby the viral genome wraps around the ejectosome within the capsid, which has never been reported before. We provide a clear, step-by-step method for the technically challenging reconstruction of ejectosomes using standard, open-source software.

microbiology↗

bioRxiv2023

Total infectome investigation of diphtheritic stomatitis in yellow-eyed penguins (Megadyptes antipodes) reveals a novel and abundant megrivirus

First identified in 2002, diphtheritic stomatitis (DS) is a devastating disease affecting yellow-eyed penguins (Megadyptes antipodes, or hoiho in te reo M[a]ori). The disease is associated with oral lesions in chicks and has caused significant morbidity and mortality. DS is widespread among yellow-eyed penguin chicks on mainland New Zealand yet appears to be absent from the subantarctic population. Corynebacterium spp. have previously been suspected as a causative agent yet, due to inconsistent cultures and inconclusive pathogenicity, its role in DS is unclear. Herein, we used a metatranscriptomic approach to identify potential causative agents of DS by revealing the presence and abundance of all viruses, bacteria, fungi and protozoa - together, the infectome. Oral and cloacal swab samples were collected from presymptomatic, symptomatic and recovered chicks along with a control group of healthy adults. Two novel viruses from the Picornaviridae were identified, one of which - yellow-eyed penguin megrivirus - was highly abundant in chicks irrespective of health status but not detected in healthy adults. Tissue from biopsied oral lesions also tested positive for the novel megrivirus upon PCR. We found no overall clustering among bacteria, protozoa and fungi communities at the genus level across samples, although Paraclostridium bifermentans was significantly more abundant in oral microbiota of symptomatic chicks compared to other groups. The detection of a novel and highly abundant megrivirus has sparked a new line of enquiry to investigate its potential association with DS.

microbiology↗

bioRxiv2022

A novel gyrovirus associated with a fatal respiratory disease in yellow-eyed penguin (Megadyptes antipodes) chicks

Yellow-eyed penguins (Megadyptes antipodes), or hoiho in te reo M[a]ori, are predicted to become extinct on mainland Aotearoa New Zealand in the next few decades, with infectious disease a significant contributor to their decline. A recent disease phenomenon termed respiratory distress syndrome (RDS) causing lung pathology has been identified in very young chicks. To date, no causative pathogens for RDS have been identified. In 2020 and 2021, the number of chick deaths from suspected RDS increased four- and five-fold, respectively, with a mortality rate of >90%. Here, we aimed to identify possible pathogens responsible for RDS disease impacting yelloweyed penguins. Total RNA was extracted from tissue samples collected during post-mortem of 43 chicks and subject to metatranscriptomic sequencing. From these data we identified a novel and highly abundant gyrovirus in 80% of tissue samples. This virus exhibited only 41% amino acid identity within VP1 to its closest relative, Gyrovirus 8, discovered in a diseased seabird. No other exogenous viral transcripts, nor pathogenic bacterial, protozoal and fungal organisms, were identified in these tissues. Due to the high relative abundance of viral reads, it is likely that this novel gyrovirus is associated with RDS in yellow-eyed penguin chicks. Author SummaryNew Zealands population of yellow-eyed penguins, also called hoiho, are predicted to become extinct in the next 20-30 years, with disease a major factor contributing to their decline. A new disease, causing fluid and bleeding into the lungs, was initially identified in 2019 in very young chicks. It was characterised as causing respiratory distress with a mortality of >90% usually within the first week of life. To date, no causative pathogens of the disease have been identified. We aimed to identify possible pathogens responsible for respiratory disease in these penguin chicks. A metatranscriptomic survey of dead chicks identified a novel and highly abundant gyrovirus present in diseased tissue, with closely related viruses causing disease in other avian hosts. It is, therefore, highly likely that this novel gyrovirus is associated with respiratory disease in these chicks. This finding offers the potential to increase the success of disease management in the critically endangered yellow-eyed penguin and possibly other at-risk penguin species. The potential to lessen mortality and slow the decline of the species is essential in protecting the biodiversity of New Zealands fauna and flora.

microbiology↗

bioRxiv2022

A lipopolysaccharide-dependent phage infects a pseudomonad phytopathogen and can evolve to evade phage resistance

Bacterial pathogens are major causes of crop diseases, leading to significant production losses. For instance, kiwifruit canker, caused by the phytopathogen Pseudomonas syringae pv. actinidiae (Psa), has posed a global challenge to kiwifruit production. Treatment with copper and antibiotics, whilst initially effective, is leading to the rise of bacterial resistance, requiring new biocontrol approaches. Previously, we isolated a group of closely related Psa phages with biocontrol potential, which represent environmentally sustainable antimicrobials. However, their deployment as antimicrobials requires further insight into their properties and infection strategy. Here, we provide an in-depth examination of the genome of {Phi}Psa374-like phages and show that they use lipopolysaccharides (LPS) as their main receptor. Through proteomics and cryo-electron microscopy of {Phi}Psa374, we revealed the structural proteome and that this phage possess a T=9 capsid triangulation, unusual for myoviruses. Furthermore, we show that {Phi}Psa374 phage resistance arises in planta through mutations in a glycosyltransferase involved in LPS synthesis. Lastly, through in vitro evolution experiments we showed that phage-resistance is overcome by mutations in a tail fiber and structural protein of unknown function in {Phi}Psa374. This study provides new insight into the properties of {Phi}Psa374-like phages that informs their use as antimicrobials against Psa. Originality-Significance StatementThe rise of phytopathogen resistance to agrichemicals poses a significant threat to crop production, and requires urgent attention. The work presented here examines a phage genus, members of which utilize LPS as a receptor, and show potential as biocontrol agents of Psa. Studies in planta showed the development of Psa resistance to the phage, whilst maintaining bacterial virulence. This finding underscores the importance of examining phages in their ecological context for the informed design of phage cocktails that are effective in phytopathogen control.

microbiology↗