Feature: Inside the bacterial machine

For something that has been on the world evolutionary stage as long as bacteria, there is certainly a lot about them that we ‘higher’ beings do not know. This is especially important considering that, despite our best efforts, some of these bugs continue to punch way above their weight on the human disease scale.

Professor Trevor Lithgow and his team at Monash University in Melbourne are keen to address this knowledge imbalance by exploring the more fundamental aspects of bacterial cell biology, and in the process perhaps uncover new ways to fight bacterial disease.

Lithgow’s group works on protein targeting, looking at how proteins are transported to their correct location inside and outside of cells, and how secreted proteins catch a ride from where they are made inside the bacterial cell to the outer membrane.

Bacteria have evolved diverse mechanisms for protein transport across membranes for all sorts of functions, and in the case of the outer membrane of Gram-negative bacterial pathogens, such proteins are often directly involved in causing disease. However, despite their obvious importance as potential anti-bacterial drug targets, these processes remain quite poorly understood.

At the Hunter Meeting in March, Lithgow revealed an exciting story in this protein transport genre, a story that is likely to hit the journals soon. As well as adding a huge chunk of clarity to the soup of bacterial protein secretion, the findings also stand to provide a brand new and tantalising drug target for bacterial disease.

Once upon a time

According to Lithgow it all started about eight years ago with some stellar work by Jan Tommassen’s lab in the Netherlands that finally identified a key part of the outer membrane protein (OMP) assembly machinery in bacteria.

The central component of this machinery was a protein called Omp85 or BamA, and Thommassen demonstrated that mutant bacteria in which the function of BamA is ‘turned down’ lose their ability to assemble proteins into their outer membrane. The protein was also essential for cell viability, because if the BamA gene was knocked out completely, the mutant bacteria died.

This was a big thing in the field, says Lithgow, because it came after the who’s who of bacteriology had spent around 20 years looking for the mechanism by which outer membranes are built and maintained in bacteria.

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“Finding this out matters a lot because the outer membrane is literally the permeability barrier for bacterial pathogens, at least for all gram-negatives – it controls how bacteria are affected by antibiotics and how they take in the nutrients they need to survive,” he says.

“We were also particularly excited at the time, because as soon as the Omp85/BamA findings came out, we used some fairly nifty bioinformatics to reveal a version of this same protein (a homologue) as part of the mitochondria in yeast and human cells. We then showed experimentally that our Omp85 equivalent is indispensable for making the mitochondrial outer membrane.

“So, as an aside, we uncovered this beautiful evolutionary link whereby mitochondria, which evolved from bacteria in the first place, kept the original mechanism used to make an outer membrane with just a few modifications to make it work in a eukaryotic cell. Really very cool,” he says.

In fact, Lithgow and his team continue to probe this story of evolution that could reveal important biological data on both the basic science and clinically relevant fronts.

We now know that BamA is an essential and highly conserved member of the Omp85 protein super-family and that all bacteria with outer membranes express BamA. It is also established that Omp85 family members all function in protein translocation through and/or assembly into cellular membranes.

Not long after this work, Lithgow also started to play with the bacterial Omp85/BamA protein, including using bioinformatics to ask how many similar proteins actually occur in bacteria and what might they do.

Addressing such questions was becoming increasingly easier with the ever-expanding number of bacterial genome sequences available. And so, without even going into the lab, Lithgow’s team charted at least two other promising groups of bacterial Omp85 proteins.

One of these was already known to work in bacteria membrane transport as a component of the two-partner secretion system (often referred to as T5SSb proteins). But it was the other protein find that got all their hearts racing. Named TamA, this protein appeared to be widespread throughout the gram-negative bacterial world.

It showed close sequence similarity to known outer membrane transporter BamA and, best of all, nobody knew anything about it. This was clearly a job for a PhD student!

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Hard yards yield reward

So, Lithgow looked to his newest student, Joel Selkrig, to find out just what this TamA thing does, and four years on it is all good news. “It is never easy to find the function of an unknown protein,” says Lithgow. “Joel did a fantastic job to overcome several technical challenges and get where he did – and this paper is the result.”

As research projects invariably do, addressing the basic question of what TamA does led Selkrig and the Lithgow group down many unexpected paths and to some bonus pots of gold.

One such bonus finding was that TamA is a component part of a novel transport system in bacteria, which they called the TAM (translocation and assembly module). This is a complex of interacting proteins embedded in both membranes of gram-negative bacteria cell walls. They then sought to reveal the details of its function.

“Infection studies using a mouse model developed by Liz Hartland’s lab at the University of Melbourne showed that bacterial mutants lacking the TAM were inferior at causing disease, inferring a role for the TAM machinery in bacterial pathogenesis,” says Lithgow.

Then, while further characterising the TAM-deficient bacteria, Selkrig found something that he immediately knew was important. He noticed that major proteins were missing from the outer membrane of the mutants and that this explained the several virulence defects.

Thus, in the course of characterising the TamA protein, a ‘new’ general mechanism for protein secretion had been discovered, mediated through the TAM complex.

“We are currently working to define how big a set of protein substrates use the TAM to get out of bacteria, to understand exactly what this molecular machine is capable of, and how it works. The methods that we’ve used in the past to characterise mitochondrial protein targeting machines work brilliantly on bacteria, so real headway is being made.

A promising and sustainable drug target

“We are also quite excited about the prospect of TamA as a druggable target in bacteria,” says Lithgow. “If you can block virulence factor transport in pathogenic bacteria, you will reduce bacterial virulence.

So if you could knock out the TAM function using something like a small molecule or antibody, and thereby knock out secretion of a whole range of virulence factors, you could reduce bacterial pathogenicity and disease.”

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The very nifty and attractive thing about this potential target is that knocking out the TAM would severely affect bacterial fitness in terms of disease, without affecting the bacteria’s ability to grow and survive as a population. Therefore, unlike the case with most antibiotics, there would be minimal selective pressure on the bacteria to overcome the drug’s effect and develop resistance mechanisms.

This approach accords with the current thinking amongst microbiologists, says Lithgow. “Most of the available antibiotics have such propensity to promote drug resistance because they are often affecting some core feature of the bacterium, and that drives a huge selective pressure to start growing and making proteins again.”

But bacteria do not need to cause disease – they are just trying to find a nice warm cosy spot to settle down and have a family. “We really need drug targets whereby we could stop disease, without promoting the same sort of selective pressure for resistance, and the cool thing for us now is that the TAM is exactly one of those sorts of targets.”

One longish-term plan for Lithgow on the bacterial front is to find small molecules that inhibit the TAM function and thereby autotransporter secretion. He has recently embarked on an exploratory study in collaboration with a drug design group at the Monash Institute of Pharmaceutical Sciences.

“We are doing some proof of principle work, looking for small molecules that would interact with the TAM machinery. We would then test if these agents can affect protein secretion, at least in a petri dish. The larger question in that domain will be for someone else to pick up, which is what we hope to come out of the publication.”

Making the most of new opportunities

About three years ago, and after many years establishing and nurturing his own niche in mitochondrial cell biology at the University of Melbourne, Trevor Lithgow moved across town to Monash University to take up an ARC Federation Fellowship.

Obtaining this prestigious five-year fellowship often means renewed academic freedom for the recipient, but it can also present many opportunities to employers and collaborators in terms of doing ‘something bold.’

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And so it was for Lithgow and Monash, with the venture built on strategic design that included establishing a brand new research Unit for Host-Pathogen Molecular Biology that would complement his own research activities.

The unit quickly grew into six groups and, as an extra challenge, all the group leaders except Lithgow were scientists establishing their very first independent research lab.

“This has obviously been very challenging,” says Lithgow, “with five people all trying to set up and do things like get students and start-up funding all at the same time. But there has been such a spirit of cooperation and a whole lot of enthusiasm to be part of a new and carefully planned ‘entity,’ and it has actually worked out as I hoped.

“The personalities and ideas involved have made it work, and in the end I see that we have been able to do much better and more strategic science because we had the luxury of choosing only the best ideas to proceed with from the start.”

The move to Monash also meant a bit of a scientific shift for Lithgow and his team. In addition to their long-standing and very successful focus on the protein comings and goings of the mitochondrial organelle, they suddenly had the right expertise, collaborations and facilities around them to really expand their bacterial protein transport focus on a larger scale than would otherwise have been possible.

Indeed, Lithgow was delighted that their interests, techniques and expertise in mitochondria have translated almost directly across to the bug-eat-bug world of the host-pathogen unit with very few teething problems. “My lab is now about half-half mitochondria and bacteria.

“This particular story about the TAM is a really nice example of the sort of things we wanted to be able to do in the original design of the unit, and to see it come to fruition after a couple of years of doing the really hard yards is very satisfying.

“This year, several other stories will break from the other labs in the unit too. Finally, it is evidence that the whole venture was worthwhile for all of us in the unit, for Monash and importantly, for the funding bodies supporting it.”