Role of Type IV pilus retraction in bacterial pathogenesis – RETRACTOPATH

Aim 1. To determine the impact of pilus retraction on meningococci
Using comparative transcriptomic and metabolomic approaches to compare the responses of wild type and retraction-defective bacteria to mechanical stress in vitro and infection in vivo, we will identify key bacterial effectors regulated by Tfp retraction and address their role in meningococcal pathogenesis.

Aim 2. To determine the impact of pilus retraction on vascular cells
Based on preliminary data, we will dissect the role of the endothelial receptor PECAM-1 and associated partners as mechanosensors for Tfp retraction and we will fully characterize the genes/proteins events/signaling pathways induced by Tfp retraction and their involvement in vascular alteration.

Aim 3. To determine the impact of pilus retraction in meningococcal pathogenesis in vivo
Using comparative transcriptomic and metabolomic approaches to analyze the host responses in vivo to infection by wild type and retraction-defective bacteria, we will identify the cellular factors activated upon Tfp retraction and will address their role in the pathogenesis of N. meningitidis.

We have shown that meningococcal strain unable to retract Tfp (?pilT) induces in vivo the formation of vascular lesions associated with intravascular coagulation and an overwhelming inflammation, similar to what is observed with the wild type strain. However, this strain is unable to promote a sustained bacteremia and lethality in mice. This data demonstrate that Tfp retraction plays a key role in the pathogenesis and outcome of meningococcal sepsis (Barnier et al, PloS Pathogen 2021).

Aim 1. To determine the impact of pilus retraction on meningococci
All the omic data have been collected and both the proteomic and metabolic profiling data are being cross-compared with the gene signature expression obtained by RNA-Seq analyses on mirror samples.
With the Dual RNA seq approach, we have already identified two bacterial hits, differentially regulated in WT and ?pilT strains in vivo, and associated with enhanced pathogeny. Now, we are characterizing their effects and mode of action both in vitro and in vivo.

Aim 2. To determine the impact of pilus retraction on vascular cells
Following our preliminary data, we have investigated and confirmed the role of PECAM-1 as a mechanical sensor for Tfp retraction. Now, we are performing the biophysical approaches and we further characterize the PECAM-1-associated signaling cascades that contribute to vascular alteration.

Aim 3. To determine the impact of pilus retraction in meningococcal pathogenesis in vivo
By conducting a comparative RNAseq analysis on non-infected and infected human skin grafts we revealed 1/ the host genes regulated upon infection with both WT and ?pilT strains, corresponding to factors promoting thrombosis, vascular leakages, inflammation; and 2) the host genes regulated solely by the WT strain, hence potentially involved in a lethal sepsis. Now, we aim to characterize the role of these genes in meningococcal pathogenesis.

We are at the halfway point of this project. Our perspectives are to achieve our different objectives.
This project will deepen our knowledge of the factors induced in meningococci upon Tfp retraction and provide a global overview of the mechanosensitive host response that ultimately leads to death.

Type IV pilus retraction enables sustained bacteremia and plays a key role in the outcome of meningococcal sepsis in a humanized mouse model.
Barnier JP, Euphrasie D, Join-Lambert O, Audry M, Schonherr-Hellec S, Schmitt T, Bourdoulous S, Coureuil M, Nassif N, El Behi M.
PLoS Pathog (2021)17(2): e1009299.

Meningococcal disease: A paradigm of type-IV pilus dependent pathogenesis.
Dos Santos Souza I, Maïssa N, Ziveri J, Morand PC, Coureuil M, Nassif X, Bourdoulous S.
Cell Microbiol. (2020) 22:e13185. Review.

Strategies used by bacterial pathogens to cross the blood–brain barrier.
Le Guennec L, Coureuil M, Nassif X, Bourdoulous S.
Cell Microbiol. (2020) 22:e13132. Review.

Molecular interactions between Neisseria meningitidis and its human host.
Coureuil M, Jamet A, Bille E, Lécuyer H, Bourdoulous S, Nassif X.
Cell Microbiol. (2019) 21(11):e13063. Review.

Type IV pili (Tfp) are long filamentous structures that are produced by numerous pathogenic bacteria and the only filament present in both Gram negative and Gram positive bacteria. They mediate an extraordinary array of functions, including motility, DNA uptake, bacterial aggregation or adherence to abiotic surface or host cells. They are highly dynamic, as they are able to rapidly retract and elongate, a process that has been linked to twitching motility. The dynamic extension/retraction cycle of Tfp centers on two cytoplasmic ATPases with antagonistic functions and that are highly conserved among Tfp-expressing species, implicating a fundamentally conserved mechanism of Tfp biology. Pilus retraction generates remarkably high forces (exceeding 100 pN), making the Tfp system among the strongest molecular motors known. There is now growing evidences that force generated by Tfp retraction act as a mechanical signal to both bacteria and host cells. Bacteria have evolved a wide range of sensing systems that can measure the mechanically induced conformational changes in stretched Tfp to appropriately respond to environmental signals and regulate the transcription of key virulence factors. While on the host side, Tfp retraction forces can induce cytoskeletal reorganization, activation of stress-activated pathways or cytoprotective signaling. Paradoxically, even though numerous bacterial pathogens generate retraction forces, to date how such forces are sensed in host cells and contribute to bacterial pathogenesis has been totally unexplored.
Using Neisseria meningitidis (meningococcus) as a model organism, as it interacts with vascular cells via its Tfp and promotes the formation of purpuric lesions often evolving towards a fatal outcome, we found, in relevant in vitro and in vivo models of infection, that pilus retraction is required to promote endothelial dysfunctions and animal death. We hypothesize that Tfp retraction (i) potentiates bacterial virulence and/or (ii) triggers deleterious mechano-sensitive pathways within the host.

The overall objective of this proposal is therefore to decipher the role of Tfp retraction in meningococcal pathogenesis and to determine how this influences the outcome of infection. We will pursue 3 major aims:

Aim 1. To determine the impact of pilus retraction on meningococci
Using comparative transcriptomic and metabolomic approaches to compare the responses of wild type and retraction-defective bacteria to mechanical stress in vitro and infection in vivo, we will identify key bacterial effectors regulated by Tfp retraction and address their role in meningococcal pathogenesis.

Aim 2. To determine the impact of pilus retraction on vascular cells
Based on preliminary data, we will dissect the role of the endothelial receptor PECAM-1 and associated partners as mechanosensors for Tfp retraction and we will fully characterize the genes/proteins events/signaling pathways induced by Tfp retraction and their involvement in vascular alteration.

Aim 3. To determine the impact of pilus retraction in meningococcal pathogenesis in vivo
Using comparative transcriptomic and metabolomic approaches to analyze the host responses in vivo to infection by wild type and retraction-defective bacteria, we will identify the cellular factors activated upon Tfp retraction and will address their role in the pathogenesis of N. meningitidis.

This project will deepen our knowledge of the factors induced in meningococci in vitro and in vivo upon Tfp retraction and provide a global overview of the mechanosensitive host response that ultimately leads to animal death.