Threading for spreading

Threading for spreading

The free-living soil nematodes, including the model organism Caenorhabditis elegans, offer a unique opportunity to study the host-pathogen interactions. Generally lacking professional innate immune cells, their epithelia, including the hypodermis and intestinal cells, also serve as defense barriers against the myriad of bacterial pathogens that may also be present in their food source. Thanks to their transparency, the course of infection in these tissues, and even the deeper layer like body-wall muscle, can be readily observed in vivo with microscopy. This feature allows the discovery of host-pathogen interactions that may be overlooked in other systems. Uncovering these novel mechanisms is the long-standing interest of our lab, and we use wild-caught nematodes and their associated microbes to dissect these interactions. 

When I first joined the lab, I was working on a project in which we and our collaborators were characterizing a collection of microsporidia (fungal-like parasites) infecting different tissues in different hosts for comparative studies. Among the strains we isolated from ecological samples was an Oscheius tipulae isolate containing what was thought to be a microsporidian inside intestinal cells. But it could not be identified using microsporidian probes or ribosomal RNA amplification. So, we decided to tackle the problem more systematically. Visually, the microbe under Nomarski microscopy was too large to be a virus but too small for other eukaryotic parasites known up to that point in nematodes, such as fungi or oomycetes. Therefore, it seemed the most likely identity was a bacterium. Luckily, we had a universal 16s ribosomal RNA probe to bacteria in the lab. We conducted in situ hybridization with this probe on the worms and saw them light up with green signals in the dark of the microscope room. This was an “ah ha, gotcha!” moment for us. We quickly realized what this meant, as we had discovered the first intracellular bacterial pathogen in free-living nematodes. However, we had a second, almost immediate “ah ha” moment when we saw many infected animals with long filaments running along their anterior-posterior axes.

Forms dictate functions. That has been one of the main paradigms governing biology. Being new to the bacteriology world, my advisor and I simply leaned on that paradigm and drew a parallel to what we know about the filamentous form of the other, more familiar eukaryotes – fungi. We thought the bacterial filaments must serve the same purpose as the hyphae of fungi, facilitating the spreading of the bacteria in the host. We assumed that this must have been recorded in the literature for other bacteria. However, to our surprise, multiple searches with key words “bacterial filaments” and “intracellular spreading” did not show any prior observations of intracellular bacterial filaments used for spreading or inside multiple neighboring host cells. At that point, my advisor decided to formally posit the most obvious hypothesis based on our observations, that filamentation by this bacterial pathogen is used for cell-to-cell spreading. Grasping the novelty of this project, he immediately wanted me to work full-time on this bacterial pathogen. And so, my PhD journey took an unexpected turn, moving away from microsporidia and venturing into the field of bacterial physiology.

The long bacterial filaments lethal to Oscheius tipulae are reminiscent of the thread of life cut by the Greek Fate Atropos when one dies, inspiring the name Bordetella atropi 

Among the first experiments that we established as vital to the project was demonstrating the intracellular nature of the bacteria. Through TEM, we were able to detect the presence of B. atropi filaments inside the cytoplasm of the host cells. However, we knew that we had to show this characteristic of the bacteria in multiple ways, and it was clear to us that colocalization of the bacteria with some cytoplasm marker would be an orthogonal approach to the TEM. Unfortunately, O. tipulae has not been as easily amenable to transgenesis as its cousin C. elegans, so we had to rely on chemical dyes. Knowing the difficulty of delivering chemical compounds in nematodes, especially in fixed animals, I focused on live cell-tracker dyes with small molecular structures. By testing an array of dyes that can be conjugated into intracellular proteins, we eventually succeeded in staining the cytoplasm of infected host intestinal cells. This allowed us to find that the presence of bacteria resulted in the absence of stained cytoplasmic materials, bolstering the notion that the bacteria indeed reside inside host cells.

Another key experiment was to alter the filamentation capacity of the bacteria in vivo so we could test the hypothesis that filamentation was required for spreading in the host. The small white board in the corner of our lab was soon decorated with color-inked drawings, outlining ideas and strategies to tackle the task. After a few hours of brainstorming, we ended up with a simple, low-tech approach based on the knowledge that a related species of our bacteria, the B. avium, has been reported to form filaments in a rich growth medium, and that b-lactam antibiotics are commonly used to induce filamentation in E. coli. Through multiple rounds of in vitro filamentation induction and filtering through 5-mm membranes to select for the non-filamenting mutants, we were eventually able to isolate a mutant deficient in cell-to-cell spreading. Our third “ah ha” moment came when we conducted whole genome sequencing and identified that the mutant carried a SNP in a well-studied bacterial metabolic enzyme GtaB. When we found out that the gtaB gene and its orthologs have been demonstrated to regulate cell size in rich culture media in B. subtilis and E. coli by the Levin group at WUSTL, we started to see the pieces of the jigsaw puzzle falling into place. It appeared to us that B. atropi, upon entering a host cell, detects the nutrient-rich environment and initiates filamentation to spread through multiple host cells.

Our study indicates that B. atropi, perhaps through co-evolution with the host, has repurposed a highly conserved metabolic pathway governing bacterial cell size to maximize its reach to other host cells. We hope that this study will spur investigations in other bacterial pathogens to see if they can also use filamentation as a cell-to-cell spreading mechanism or use the same metabolic pathway to trigger the phenotype in vivo.