Institute of Fundamental Technological Research
Polish Academy of Sciences

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Liam Feltham


Doctoral thesis
2022-01-01 Using live cell imaging to study interactions between Listeria monocytogenes and host cells during infection 
supervisor -- Paweł Paszek, PhD, DSc, IPPT PAN
 

Recent publications
1.  Feltham L., Moran J., Goldrick M., Lord E., Spiller D. G., Cavet J. S., Muldoon M., Roberts I. S., Paszek P., Bacterial aggregation facilitates internalin-mediated invasion of Listeria monocytogenes, Frontiers in Cellular and Infection Microbiology, ISSN: 2235-2988, DOI: 10.3389/fcimb.2024.1411124, Vol.14, pp.1411124-01-18, 2024

Abstract:
Dissemination of food-borne L. monocytogenes in the host relies on internalin-mediated invasion, but the underlying invasion strategies remain elusive. Here we use live-cell microscopy to follow single cell interactions between individual human cells and L. monocytogenes and elucidate mechanisms associated with internalin B (InlB)-mediated invasion. We demonstrate that whilst a replicative invasion of nonphagocytic cells is a rare event even at high multiplicities of invasion, L. monocytogenes overcomes this by utilising a strategy relaying on PrfA-mediated ActA-based aggregation. We show that L. monocytogenes forms aggregates in extracellular host cell environment, which promote approximately 5-fold more host cell adhesions than the non-aggregating actA-ΔC mutant (which lacks the C-terminus coding region), with the adhering bacteria inducing 3-fold more intracellular invasions. Aggregation is associated with robust MET tyrosine kinase receptor clustering in the host cells, a hallmark of InlB-mediated invasion, something not observed with the actA-ΔC mutant. Finally, we show via RNA-seq analyses that aggregation involves a global adaptive response to host cell environment (including iron depletion), resulting in metabolic changes in L. monocytogenes and upregulation of the PrfA virulence regulon. Overall, our analyses provide new mechanistic insights into internalin-mediated host-pathogen interactions of L. monocytogenes.

Keywords:
Listeria monocytogenes, host-pathogen interactions, aggregation, PrfA regulon, livecell microscopy

Affiliations:
Feltham L. - other affiliation
Moran J. - other affiliation
Goldrick M. - other affiliation
Lord E. - other affiliation
Spiller D. G. - other affiliation
Cavet J. S. - other affiliation
Muldoon M. - other affiliation
Roberts I. S. - other affiliation
Paszek P. - IPPT PAN
2.  Moran J., Feltham L., Bagnall J., Goldrick M., Lord E., Nettleton C., Spiller David G., Roberts I., Paszek P., Live-cell imaging reveals single-cell and population-level infection strategies of Listeria monocytogenes in macrophages, Frontiers in Immunology, ISSN: 1664-3224, DOI: 10.3389/fimmu.2023.1235675, Vol.14, pp.1235675-1-17, 2023

Abstract:
Pathogens have developed intricate strategies to overcome the host’s innate immune responses. In this paper we use live-cell microscopy with a single bacterium resolution to follow in real time interactions between the food-borne pathogen L. monocytogenes and host macrophages, a key event controlling the infection in vivo. We demonstrate that infection results in heterogeneous outcomes, with only a subset of bacteria able to establish a replicative invasion of macrophages. The fate of individual bacteria in the same host cell was independent from the host cell and non-cooperative, being independent from co-infecting bacteria. A higher multiplicity of infection resulted in a reduced probability of replication of the overall bacterial population. By use of internalisation assays and conditional probabilities to mathematically describe the two-stage invasion process, we demonstrate that the higher MOI compromises the ability of macrophages to phagocytose bacteria. We found that the rate of phagocytosis is mediated via the secreted Listeriolysin toxin (LLO), while the probability of replication of intracellular bacteria remained constant. Using strains expressing fluorescent reporters to follow transcription of either the LLO-encoding hly or actA genes, we show that replicative bacteria exhibited higher PrfA regulon expression in comparison to those bacteria that did not replicate, however elevated PrfA expression per se was not sufficient to increase the probability of replication. Overall, this demonstrates a new role for the population-level, but not single cell, PrfA-mediated activity to regulate outcomes of host pathogen interactions.

Keywords:
Listeria monocytogenes, macrophage, single cell heterogeneity, phagocytosis, PrfA regulon, listeriolysin

Affiliations:
Moran J. - other affiliation
Feltham L. - other affiliation
Bagnall J. - other affiliation
Goldrick M. - other affiliation
Lord E. - other affiliation
Nettleton C. - other affiliation
Spiller David G. - other affiliation
Roberts I. - other affiliation
Paszek P. - IPPT PAN

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