Good morning Mr. Amoeba, may I come in? – Or how protozoa act as Trojan horses for Campylobacter

By Jonas Waldenström

The world of microbes is measured on a different scale from the world of humans. A single drop of seawater can contain more microbial life than there are inhabitants in Sweden. And the microbes are everywhere: in the sea, in the air, in the soil, even deep in the bedrock, or in the abysses of the ocean. The smallest and most numerous inhabitants of the microbial world are viruses; so tiny we need advanced electron microscopy to see them. They are obligate parasites, high jacking the cellular machinery of other organisms for replication. The prokaryotes – bacteria and archea – come next, in an abundance of shapes and forms. In many environments bacteria constitute the bulk of the microbe world in terms of mass. Next step up on the size ladder are the protozoa: a taxonomically diverse collection of single-celled eukaryotes that include amoebae, rotifers, foraminifers and many, many more bizarre creatures. The roles they play differ depending on species and circumstances. For instance, in marine systems protozoa drive photosynthesis, producing carbohydrates from sunlight. They can also be the bad guys, the trolls and ogres of the microbe world feeding on the smaller members.

Single-celled eukaryotes were lumped together as ‘Protozoa’ in the early ‘five kingdoms of life’. In the genomic age we now know that protozoa is not a valid taxonomic group, as the members in several cases are paraphyletic. But out of conveiniance the term is still used. This particular protozoa is an Ammonia tepidia, a benthic foraminifer (image by Scott Fay, UC Berkeley, via Wikimedia Commons).

Single-celled eukaryotes were lumped together as ‘Protozoa’ in the early ‘five kingdoms of life’. In the genomic age we now know that protozoa is not a valid taxonomic group, as the members in several cases are paraphyletic. But out of conveiniance the term is still used. This particular protozoa is an Ammonia tepidia, a benthic foraminifer (image by Scott Fay, UC Berkeley, via Wikimedia Commons).

Single-celled life has ruled this planet for three billion years. They are also ruling us, at the global scale by carbon cycling and atmospheric gas exchange, down to the individual scale by living in and on our bodies, either friendly as commensals, or aggressively as pathogens. In your gut there are billions of bacteria processing the food while taking tidbits for themselves. Some of these bugs work for the common good including the host (– that’s you!), some are just there on a quick visit, and some are interfering with the other microbes or you. Recent research show how intricate the microbiome of the gut is, and how important the composition of it is for our health. The ever-decreasing costs for DNA-sequencing have sparked a new branch of microbiology research. Some believe we are the doorsteps of a revolution in medicine, while others think it is snake oil.

What all can agree on, at least, is that the potential for interactions between microorganisms is nearly endless, but that we need to start addressing them to get a hold on disease epidemiology. A bug we have worked a lot with over the years is Campylobacter jejuni. This bug, also known as the ‘chicken bug’, is a major human pathogen. It rarely kills, but it affects many, many people. A common figure says that 1% of the human population in the US is infected with C. jejuni per year. Yes, per year! If you ever have had campylobacteriosis you are likely to remember it. Profuse diarrhea, vomiting and stomach pains are unpleasant, but common symptoms. Folks get sick, stay home from work or school, and some may need to seek health care. A few percent become hospitalized, and a fraction develops sequelae including paralytic symptoms. Recent research suggests that the exposure is higher than the 1% figure. Perhaps as high as 8% of the population per year, but that many infections don’t give symptoms.

Anatomy of an amoeba (image by Pearson Scott Foresman, via Wikimedia Commons)

Anatomy of an amoeba (image by Pearson Scott Foresman, via Wikimedia Commons)

Soon ten years ago, Dr Diana Axelsson-Olsson, then a graduate student, was working with C. jejuni and amoebae in the microbiology lab at Kalmar County Hospital. The aim of the project was to investigate if campylobacters interacted with amoebae, and if that was of importance for how the bacteria fared in the environment. In those days (and to a large extent still) the focus in our lab was wild bird campylobacters, and the environment represented a ‘black box’ in the epidemiology. Diana showed that if C. jejuni were exposed to amoebae the bacteria quickly ended up inside the amoebae. In the microscope they could be seen swimming around in amoebae vacuoles. More importantly, she found that raising the temperature to 37°C the bacterial cells started to multiply inside the amoebae. In fact, they grow so quick and so violently that they soon ruptured the amoebae and spilled out into the surrounding solution!

Perhaps this is a good place to pause for a while, and tell you something about the biology of campylobacters. Doing so make it easier for you to understand the importance of this finding. C. jejuni is an obligate gastrointestinal bacterium that only replicate at temperatures of 37-42°C, in essence the body temperature range of its host animals. It is also a rather sensitive bug, that doesn’t do well in aerobic conditions, doesn’t stand UV-radiation (sunshine, that is) or desiccation very well. Compared to Salmonella it is a real softie. Despite this, C. jejuni is one of the most common pathogens, equipped with a large host range of wild birds, domestic birds and mammals, and humans. Some would say, even, that campylobacters are ubiquitous. This duality of being sensitive and ubiquitous has been a longstanding conundrum in C. jejuni research.

Thus, in light of this, the campy-within-the-amoeba was the start of something new. A potential pathway for the bacterium to survive outside its animal host. Later studies have shown that it is not only amoebae of the genus Acanthamoeba (the ones the research started with) that take up campylobacters, rather this ability seems widespread among protozoa. So, if campylobacters from an infected host end up in an aquatic environment – and we are not talking oceans here, but rather puddles – there is likelihood they will meet an amoeba. When that happens, the bacteria somehow, either actively or passively, are taken up by the amoeba and become internalized. The life within the vacuole seems fairly cozy, in fact it has been shown that once internalized the bugs can survive and be able to become resuscitated many weeks, even months, later. And, of course, the raise of temperature to 37°C is expected to take place when an animal drinks the water with the bug-infested amoebae, leading to exponential growth of the bacteria, rupture of the poor amoebae and fait-a-complit infection of the new host! A Trojan horse epidemiological pathway!

The Spartans are up for a surprise (Giovanni Domenico Tiepolo, via Wikimedia Commons)

The Spartans are up for a surprise (Giovanni Domenico Tiepolo, via Wikimedia Commons)

Such pathways are known from other systems, perhaps most famously from Vibrio cholera, the bacterium causing human cholera. Vibrio bacteria are transmitted via contaminated water, and it has been shown that bacteria that invade protozoa start to express genes that make them more virulent in humans.

An open-ended question for the Campylobacter vs. Amoeba story has been whether it is an active or passive event from the bacterium’s side. Amoebae normally eat bacteria for breakfast, lunch and dinner, stretching out their long pseopodes to grab things in their environment. Food is ingested into phagosomes that turn into lysosomes and the contents get degraded. How then do campylobacters survive this? Jenny Olofsson in our lab has devoted her PhD on this and related questions, and a few weeks ago one of her studies was published in PLOS ONE.

Viable and heat killed C. jejuni are taken up into different types of A. polyphaga vacuoles. (A) Live/Dead stained viable (green) C. jejuni, confined to tight vacuoles. (B) Live/Dead stained heat killed (red) C. jejuni residing in giant spacious vacuoles. (C) Vaculoes with CTC stained viable (red) C. jejuni do not co-localize with Alexa fluor-488 labeled dextran filled vacuoles (green). (D) Vaculoes with CTC stained heat killed (red) C. jejuni have taken up Alexa fluor-488 labeled dextran (green). (E) In contrast to non digestive vacuoles, giant digestive vacuoles contained smaller vesicles (arrow). Picture D and E are from the same amoeba. doi:10.1371/journal.pone.0078873.g004

Viable and heat killed C. jejuni are taken up into different types of A. polyphaga vacuoles.
(A) Live/Dead stained viable (green) C. jejuni, confined to tight vacuoles. (B) Live/Dead stained heat killed (red) C. jejuni residing in giant spacious vacuoles. (C) Vaculoes with CTC stained viable (red) C. jejuni do not co-localize with Alexa fluor-488 labeled dextran filled vacuoles (green). (D) Vaculoes with CTC stained heat killed (red) C. jejuni have taken up Alexa fluor-488 labeled dextran (green). (E) In contrast to non digestive vacuoles, giant digestive vacuoles contained smaller vesicles (arrow). Picture D and E are from the same amoeba.
doi:10.1371/journal.pone.0078873.g004

By comparing live and dead campylobacters and the speed of which they were taken up by amoebae, Jenny and colleagues could show that it is a process that is induced actively by the bacterium. To start with, viable bacteria associated with a substantially higher proportion of Acanthamoeba trophozoites than heat-killed bacteria. The speed of internalization, the total number of internalized bacteria as well as the intracellular localization of internalized C. jejuni were also dramatically influenced by bacterial viability. Living C. jejuni ended up in small vacuoles that were tightly surrounding the bacteria, while heat-killed C. jejuni were observed in larger, spacious vacuoles. Using fluorescently labeled dextran, it was shown that the latter vacuoles were part of the normal degradative pathway – in other words, chewed, swallowed down and digested. Somehow the live C. jejuni cells manage to escape the amoeba metabolic machinery, perhaps by stopping the fusion of the phagosome with the lysosome. The next step would be to identify the genes responsible for theses processes and characterize the molecular mechanisms involved.

Clearly, the usage of amoebae and other protozoa has many advantages for pathogenic bacteria that are poor in surviving in the environment. By infecting amoebae they can take advantage of the intracellular environment and hope that the protozoan cell lives long enough to become swallowed by a suitable host animal. Each and every water body is in essence filled with a cavalry of potential mini-Trojan horses.

Link to the articles:

Olofsson J, Axelsson-Olsson D, Brudin L, Olsen B, Ellström P (2013) Campylobacter jejuni Actively Invades the Amoeba Acanthamoeba polyphaga and Survives within Non Digestive Vacuoles. PLoS ONE 8(11): e78873. doi:10.1371/journal.pone.0078873

Axelsson-Olsson D, Waldenström J, Broman T, Olsen B, Holmberg M (2005) Protozoan Acanthamoeba polyphaga as a Potential Reservoir for Campylobacter jejuni. Appl. Environ. Microbiol. 71: 987-992. doi:10.1128/AEM.71.2.987-992.2005

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