How do bacteria sense and navigate chemical gradients within biofilms?

Most bacteria live attached to surfaces where they form dense communities called biofilms. While some of these assemblages play beneficial roles in our lives, infections within the human body are often very difficult to treat because biofilms protect cells from both antibiotics and the immune system. Understanding the fundamental processes that contribute to biofilm formation is essential to developing new ways to disrupt or manipulate these important bacterial communities.

The bacterial species Pseudomonas aeruginosa, which causes dangerous infections in burn victims and cystic fibrosis patients, use tiny grappling hook-like appendages called pili move within biofilms. Our group recently demonstrated that single P. aeruginosa cells can use pili-based motility to navigate to more favourable nutrient environments within a developing biofilm. We found that this process, called chemotaxis, arises because cells pull themselves in the opposite direction when they detect they are moving away from a nutrient source. This remarkable ability likely gives chemotactic cells an advantage in biofilms and opens a new way to control biofilm formation. While other forms of microbial chemotaxis have been extensively studied, relatively little is known about pili-based chemotaxis. This study will address two major gaps in our knowledge:

First, we do not know how bacteria actually sense whether they are going towards or away from the source of chemoattractant. In general, there are two different possibilities: cells could either move from one location to another and measure the change in concentration over time (temporal sensing) or they could directly sense changes in concentration over the length of their bodies (spatial sensing). The proposed work will use a combination of novel microfluidic experiments, computer based cell tracking, and bacterial genetics to directly test these two different possibilities. Our preliminary experiments indicate that cells do not increase their probability of reversing when they experience a decrease in the concentration of chemoattractants over time, suggesting that surface attached cells do not use temporal sensing to guide chemotaxis. Future experiments will use novel bacterial strains that relocalise fluorescently labelled proteins to their opposite pole when they reverse direction, which will allow us to test if cells can sense changes in concentration over their length.

Second, our experiments indicate that cells tend to travel directly up chemical gradients, yet this observation cannot be explained by known forms of bacterial motility. Surface attached bacteria are thought to have only one behaviour in their repertoire to facilitate chemotaxis: reverse direction. However, reversals alone only allow cells to explore a one-dimensional line, so if a cell happened to land on a surface perpendicular to the gradient, it would be incapable of steering towards the nutrient source. In our preliminary work, we have discovered a new way in which surface attached cells can reorient their motility. Our computer-based image analysis software reveals that cells frequently perform somersault-like manoeuvres we call "twiddles". These reorientations occur when cells steer in either a clockwise or counter-clockwise direction over a period of minutes to hours. This study will use novel bacterial strains and cutting edge super-resolution microscopy to understand how cells generate twiddles. In addition, we will combine data obtained from tracking the movement of tens of thousands of cells with mathematical models to quantify how reversals and twiddles work together to generate chemotaxis.

Taken together, this study will provide fundamentally new understanding of how cells regulate their movement within biofilms, potentially giving us new tools to inhibit biofilm development or control the motility of cells in industrial applications.

Flagella-based chemotaxis in swimming bacteria has been extensively characterized. However, most bacteria live within biofilms where cells instead use Type IV pili to move. Recent work from my group (Oliveira, Foster & Durham, PNAS, 113, 2016) shows that single Pseudomonas aeruginosa cells use pili-based motility to generate chemotaxis in a developing biofilm and control their movement with submicron precision. This project aims to resolve the fundamental mechanisms that underlie this remarkable ability.

First, we do not know how cells know whether they are moving up or down gradients. Biofilm cells could either sense temporal changes in concentration, like swimming bacteria do, or sense how the concentration changes over the length of their bodies. We have designed novel microfluidic experiments that can distinguish these two different sensing modalities and provide deeper insights into how the underlying signal transduction system functions. Excitingly, our preliminary work suggests that biofilm cells directly sense changes in concentration over the length of their bodies.

Second, we have discovered that cells possess the ability to actively rotate their bodies on a surface, a behaviour we call "twiddling". These manoeuvres occur in mutants that lack the ability to rotate their flagella, implying that cells use pili to generate twiddles. We propose using super-resolution microscopy, along with a novel bacterial strain in which the protein that drives pili retraction has been fluorescently labelled, to resolve how cells generate twiddles. In addition, we will quantify the movement of tens of thousands of cells within chemical gradients and use this to parameterize mathematical models that will allow us to understand the role of twiddles in chemotaxis.

This study will resolve how pathogenic bacteria navigate the highly heterogeneous chemical landscapes within biofilms, which could potentially give us new ways to manipulate them to our advantage.

Bacterial biofilms impact our lives in many different ways. For example, the biofilms that line our gut provide us with numerous health benefits, while in industrial applications they are used to break down environmental pollutants into more benign forms. However, biofilms also have many negative effects. For example, biofilms result in persistent, treatment-resistant infections because they protect bacteria from antibiotics and the immune system.

This study aims to understand the fundamental mechanisms that cells within biofilms use to position themselves in more favourable chemical environments. The insights gained from this study will potentially give us new tools to rationally engineer or disrupt biofilm communities. Below we outline the impact of this work on both the clinical and industrial sectors:

1. Improved treatments of clinical infections
The model organism used in this study, Pseudomonas aeruginosa, is an opportunistic pathogen and major cause of infection in vulnerable patients, including burn wound victims and patients who suffer from cystic fibrosis (CF). Most late-stage CF patients harbour chronic P. aeruginosa infections in their lungs, which are associated with a loss of quality of life, morbidity, and mortality. The UK is home to one of the largest densities of CF prevalence in the world, with approximately 1/25 people carrying a defective copy of the CFTR gene that causes CF. The lungs of those who suffer from CF (i.e., individuals with two defective copies of CFTR) are routinely infected by bacteria from environmental sources and other patients with CF. While the immediate focus of this project is on basic science, our findings may ultimately reveal new clinical interventions for novel targets/pathways. Novel approaches for bacterial interventions are urgently needed. Earlier this year, the World Health Organization named P. aeruginosa as one of three bacterial species in greatest need of R&D for new antibiotic development.

The work proposed here will develop sophisticated new tools to quantify bacterial motility in carefully controlled chemical environments. These tools can be easily used to test if the movement of virulent strains isolated from patients differs from that of more benign ones and could lead to new insights on how biofilm motility affects pathogenesis. In addition, our results can be used to provide new insights into polymicrobial infections. Bacteria and other microorganisms secrete a diverse range of chemicals (including toxins) which then form gradients within biofilms. Our analyses provide the basis to understand how cells might bias their motility towards or away from chemicals produced by other genotypes, thereby shedding new light into how different microbes compete within clinical infections.

2. The manipulation of biofilms in biotechnology applications
Our work will reveal how movement of biofilm cells can be manipulated using chemical gradients. This information will provide the means to control where biofilms form on a surface. The ability to pattern biofilms has many possible applications in biotechnology. For example, pili-based chemotaxis could potentially be used as a tool to promote or inhibit competitive exclusion in bacterial communities. The manufacture of high value goods or degradation of environmental contaminants using bacteria often requires the coexistence of multiple genotypes because each relies on the metabolic products or secretions produced by other members of the community. While the ecology of these synthetic microbial communities often makes them difficult to maintain in well-mixed conditions, systematically patterning different genotypes on a surface would reduce local competition, making the fragile members of these communities more resistant to extinction. While our work focuses on one species, many different Gram negative bacteria are capable of twitching motility. Thus, our results could potentially be used in many applications.