Alteromonas Myovirus V22 Represents a New Genus of Marine Bacteriophages Requiring a Tail Fiber Chaperone for Host Recognition

Marine phages play a variety of critical roles in regulating the microbial composition of our oceans. Despite constituting the majority of genetic diversity within these environments, there are relatively few isolates with complete genome sequences or in-depth analyses of their host interaction mechanisms, such as characterization of their receptor binding proteins (RBPs). Here, we present the 92,760-bp genome of the Alteromonas-targeting phage V22. Genomic and morphological analyses identify V22 as a myovirus; however, due to a lack of sequence similarity to any other known myoviruses, we propose that V22 be classified as the type phage of a new Myoalterovirus genus within the Myoviridae family. V22 shows gene homology and synteny with two different subfamilies of phages infecting enterobacteria, specifically within the structural region of its genome. To improve our understanding of the V22 adsorption process, we identified putative RBPs (gp23, gp24, and gp26) and tested their ability to decorate the V22 propagation strain, Alteromonas mediterranea PT11, as recombinant green fluorescent protein (GFP)-tagged constructs. Only GFP-gp26 was capable of bacterial recognition and identified as the V22 RBP. Interestingly, production of functional GFP-gp26 required coexpression with the downstream protein gp27. GFP-gp26 could be expressed alone but was incapable of host recognition. By combining size-exclusion chromatography with fluorescence microscopy, we reveal how gp27 is not a component of the final RBP complex but instead is identified as a new type of phage-encoded intermolecular chaperone that is essential for maturation of the gp26 RBP.This work was supported by grants ‘VIREVO’ CGL2016‐76273‐P (MCI/AEI/FEDER, EU) (cofounded with FEDER funds) from the Spanish Ministerio de Ciencia e Innovación and ‘HIDRAS3’ PROMETEU/2019/009 from Generalitat Valenciana. R.G.-S. was supported by a predoctoral fellowship from the Valencian Consellería de Educació, Investigació, Cultura i Esport (ACIF/2016/050) and was also a beneficiary of the BEFPI 2019 fellowship for predoctoral stays from Generalitat Valenciana and The European Social Fund. F.R.-V. was a beneficiary of the 5top100 program of the Ministry for Science and Education of Russia

Revertant Listeria monocytogenes and conditions for their persistence as intracellular L-forms

L-forms are cell wall deficient forms of bacteria that are able to survive and multiply without a rigid cell wall. Since their discovery in 1935, numerous case studies have reported an observation or isolation of such bacterial L-forms in diseased patients or animals. They have largely been associated with in chronic or recurrent diseases, where L-forms are believed to represent a strategy that allows the bacteria to escape from eradication by the host immune system and/or antibiotic therapy in the clinical setting. However, hitherto no study has definitively established the role of the L-form state in diseases nor was the occurrence of L-forms undoubtedly demonstrated and quantified. This is in particular true for the hypothesised relevance of L-forms for relapsing infectious diseases. Several cases of recurrent listeriosis have been reported so far. Sub-typing analyses suggested that the identical Listeria monocytogenes strain persisted in the patient during these cases. In addition, Listeria L-forms have been reported to be isolated from the cerebrospinal fluid of a patient and from sheep diagnosed with listeriosis. Therefore, Listeria monocytogenes L-forms are assumed to be induced, or selected, by the antibiotic therapy and to be responsible for the persistence of the pathogen although antibiotic treatment. After termination of the treatment, L-forms are hypothesised to revert to walled state by rebuilding their cell wall. However, the triggers for this reversion back to a walled state is still not known. With the reestablishment of the cell wall, the bacteria regain their virulence properties that confer the bacteria the possibility to restart the infection process. The aim of this thesis is to investigate whether antibiotic treatment might promote the development of L-forms inside previously infected non-professional phagocytes and whether the occurring L-forms can survive and persist inside the host cells. To be able to achieve this major goal, the first step was to generate transient L-form strains, called revertant strains, that have the capability to switch back and forth between the L-form and the walled state. By adapting previously established protocols it was possible to generate revertant strains that were not impaired in growth or pathogenicity compared to their wild-type strains. Thus, the revertant strains turned out to be suited for the investigation of the development of intracellular L-forms after infection and antibiotic treatment of host cells in cell culture experiments. Confocal laser scanning microscopy analysis of cell cultures infected with L. monocytogenes revertant strains expressing Green Fluorescent Protein demonstrated the development of intracellular L. monocytogenes L-forms after an antibiotic treatment with Ampicillin. The presence and identification of L. monocytogenes L-forms was further confirmed by fluorescence in situ hybridisation using oligonucleotide probes specific for Listeria genus which allowed visualisation of intracellular L-forms in situ. In order to quantify the intracellular conversion of rods to L-form, cell cultures showing intracellular L-forms were lysed and plated on suitable media. However, the results obtained with this method were not conclusive, as growth of L-forms or reverted walled bacteria from the cell culture lysate could not be observed. Validation of the viability of L-forms occurring inside the host cells was provided by extraction of L- forms using a micro-manipulator and subsequent growth of the extracted L-forms in vitro. In conclusion, the results from this study demonstrated the development of Listeria monocytogenes L- forms inside infected eukaryotic host cells by an antibiotic treatment of the cell cultures with Ampicillin. In addition, it was proved that Listeria L-forms can survive and persist intracellularly for a couple of weeks in eukaryotic host cells. These are important findings that provide evidence for he potential role of L-forms of Listeria monocytogenes in recurrent listeriosis

Ampicillin Treatment of Intracellular Listeria monocytogenes Triggers Formation of Persistent, Drug-Resistant L-Form Cells

Listeria monocytogenes is an opportunistic intracellular pathogen causing an infection termed listeriosis. Despite the low incidence of listeriosis, the high mortality rate in individuals at risk makes this bacterium one of the most dangerous foodborne pathogens. Reports about a relapse of infection after antibiotic treatment suggest that the bacteria may be able to evade antibiotic treatment and persist as a dormant, antibiotic-tolerant subpopulation. In this study, we observed intracellular generation of antibiotic-resistant L-forms of Listeria monocytogenes following Ampicillin treatment of Listeria monocytogenes infected cells. Detection and identification of intracellular Listeria L-forms was performed by a combination of fluorescence in-situ hybridization and confocal laser scanning microscopy. Using micromanipulation, it was possible to isolate single intracellular L-form cells that following transfer into fresh medium gave rise to pure cultures. In conclusion, the results obtained here provide strong evidence that antibiotic treatment of infected host cells can induce the formation of L-forms from intracellular Listeria monocytogenes. Furthermore, our results suggest that intracellular L-forms persist inside host cells and that they represent viable bacteria, which are still able to grow and proliferate.ISSN:2235-298

A Proteogenomic Resource Enabling Integrated Analysis of Listeria Genotype-Proteotype-Phenotype Relationships

ISSN:1535-3893ISSN:1535-390

2021 Bringing Genomics Data to the Clinic Hackathon

Official repository for the Bridging Genomics Data to the Clinic Hackathon hosted virtually in January 2021 by DNAnexus, the OpenCravat Group at Johns Hopkins University, and Carnegie Mellon University Libraries