Tag: salmonella typhimurium

Human intestinal microvilli. Credit: Wellcome Library, London
Sanger Life

Great balls of cells: Using intestinal organoids to study Salmonella

DATE: 10/09/15

By Jessica Forbester

Intestinal human organoids. Credit: Jessica Forbester

Intestinal human organoids. Credit: Jessica Forbester

One of the main causes of food poisoning is Salmonella enterica. This bacterium infects cells in the intestinal epithelium that lines our gut, leading to painful stomach cramps, diarrhoea and fever. One major difficulty in studying Salmonella infection is that you can’t easily study it in people’s intestines, and recreating the intestinal epithelium in a lab setting has been notoriously tricky. However, newly developed intestinal organoids are starting to provide a solution.

Intestinal organoids are little balls of intestinal epithelium, composed of a range of intestinal cell types surrounding a hollow lumen. The organoids act as a bridge between in vivo and in vitro systems.

The intestinal epithelium is an extremely important part of the immune system, with a complex structure and range of cell types. It acts as the dividing barrier between the space inside the gut tube, called the intestinal lumen, and the underlying gut tissue.

I have been generating intestinal human organoids (iHOs) from human induced pluripotent stem cells (hIPSCs). Using a protocol established by our collaborators Ludovic Vallier and his team at the Anne McLaren Laboratory for Regenerative Medicine, I take these stem cells and expose them to different chemical signals. This drives changes in gene expression, which pushes the hIPSCs to change into more specialised cells, in a process known as differentiation. The cells change first into endoderm, and then into hindgut.

To grow the balls, I place this hindgut into a pro-intestinal culture system, with a supporting Matrigel matrix. Growth factors that promote intestinal differentiation and proliferation are added to the media and, after a few weeks, little spheroids start to form.

These balls of cells are self-sustaining and can be grown for long periods of time. They take a while to mature and form structures recognisable as adult cells but, after a few months of intensive culturing, I get intestinal human organoids that are ready to work with. They can then be used as an infection model for enteric pathogens such as Salmonella.

Salmonella enterica serovar Typhimurium causes a self-limiting gastroenteritis in healthy individuals. We wanted to use intestinal human organoids to show the early interactions between S. Typhimurium and the organoids generated from a representative hIPSC-line called A1ATD-1.

Firstly, we had to ensure that the organoids contained different cell types normally found in the intestinal tract, such as Goblet cells and Paneth cells. Specific cell type markers were detected with RT-qPCR, and immunostaining showed the localisation of these markers within different groups of cells. Using transmission electron microscopy we could see clear polarisation of the cells, microvilli and tight junctions, confirming we were growing organoids that displayed characteristics of human intestinal epithelium.

S. Typhimurium would normally interact with the epithelial cells at the luminal side. The bacteria therefore needed to be delivered directly into the luminal cavity of the organoids; the centre of the sphere. This required multiple microinjections of the bacteria into the iHOs before we could collect enough RNA for RNA-Sequencing. After sequencing, we saw previously well-described responses to S. Typhimurium such as up-regulation of proinflammatory cytokines. However, genes such as BIRC3 and IL-20, whose role in Salmonella infection is not well understood, were also flagged. Interestingly, BIRC3 protein is also expressed in enteroendocrine cells, which may shed light on their role in response to intestinal infection.

Many people here at the Sanger Institute are generating human induced pluripotent stem cells so we have a large pool of genotypes to select from. We can therefore utilise our iHO system to help understand how the host genotype can alter the response of the host intestinal epithelial cells. This is a really exciting prospect as this response is crucial to the outcome of an infection, and this work provides support for using iHOs as a tool to study host-pathogen interactions at the intestinal interface.

Jessica Forbester is a PhD student at the Wellcome Trust Sanger Institute, working under the supervision of Gordon Dougan.

References:

Forbester et al. (2015). Interaction of Salmonella enterica serovar Typhimurium with intestinal organoids derived from human induced pluripotent stem cells. Infection and ImmunityDOI:10.1128/IAI.00161-15

Hannan et al. (2013). Generation of multipotent foregut stem cells from human pluripotent stem cells. Stem Cell Reports. DOI:10.1016/j.stemcr.2013.09.003

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Salmonella Typhimurium. Credit: David Goulding, Wellcome Images
Sanger Science

The bacteria that’s as smart as a whip

30 March 2015
By Fernanda Schreiber

Hj bacteria induce massive ruffles on the membrane of the host’s cells, which could explain their increased ability to invade. Credit: Fernanda Schreiber and David Goudling.

Hj bacteria induce massive ruffles on the membrane of the host’s cells, which could explain their increased ability to invade. Credit: Fernanda Schreiber and David Goudling.

Flagella are noodle-like surface structures that bacteria use to move and to attach to cells in their hosts. Salmonella typhi, the bacterium that causes typhoid fever, has three different types of flagella. However, while one can be found all over the world, the other two variants are confined to Indonesia.

Why? What kind of advantage is obtained by having a particular type of flagellin? That was the question that kicked off my PhD.

Typhoid fever, which is caused by the ingestion of food or water contaminated with Salmonella enterica serovar Typhi (S. Typhi), remains an important health threat in lower-income countries with poor sanitation. Many travellers know about it, as they need to get vaccinated if they are going to exotic places.

The principal component of the flagellar filament is a protein called flagellin. This protein is used for Salmonella classification, with most S. Typhi strains producing Hd. However, isolates from Indonesia, a country with high incidence of typhoid, show alternative flagellins: Hj and Hz66. Hj arises from a naturally occurring deletion in the Hd fliC gene. Hz66, on the other hand, is encoded by a different gene, fljB, located on a plasmid.

Using strains of S. Typhi that had been modified to express the different flagellin variants, we looked at the structure of the flagellum and the ability of the bacteria to invade host cells.

Flagella from Hj bacteria were shorter and thinner than the other two variants, but that did not impair their ability to move around. In fact, Hj bacteria were more motile and they were more effective at invading epithelial cells. We thought this might be because the filaments of Hj were better at attaching themselves to cells. However, bacteria we had genetically modified without flagella could stick to cells just as well as their hairy counterparts.

We noticed that Hj bacteria induced massive ruffles on the membrane of cells, which could explain their increased ability to invade. When we infected cells lacking key genes responsible for ruffle formation with Hj, ruffles still formed. This leads us to suspect that Hj bacteria could be exploiting alternative signals to produce ruffles that other Salmonella do not use.

Hd expressing S. Typhi were better at invading macrophages, a type of white blood cell that engulfs and digests foreign substances, which could partly explain their worldwide success. However, additional factors such as host genetics and environmental factors may play an even greater role.

Looking at samples from typhoid patients infected with strains expressing Hj or Hz66 flagellins, we found that they were older than those infected with the more common Hd strains and they had anti-Hd antibodies. This suggests that those individuals had a prior encounter with an Hd strain, symptomatic or not, and that Hj and Hz66 strains are behaving opportunistically.

Could this explain why these two strains appear only in Indonesia? If people are infected with the common strain and develop antibodies to fight it, Hd expressing S. Typhi can no longer infect them. As the number of people infected in Indonesia is so high, the bacteria in this area may have been forced to mutate to ensure they could still get round their hosts’ defences. The Hj and Hz66 strains may be taking the opportunity to re-infect those who would previously have been immune.

This is an important consideration for those designing vaccines in this area, as protection from Hd strains may just make life easier for the Hj and Hz66 strains to thrive.

Fernanda Schreiber is a Posdoctoral Fellow working as part of the CTTV team on the development of intestinal organoids as models for IBD research. She completed her PhD in Gordon Dougan’s lab on host-pathogen interactions, in particular the role of flagella during Salmonella Typhi infection. After her PhD, Fernanda worked in the Cardiovascular Division in Addenbrooks looking at the effect of cholesterol loading in macrophages.

References

  • Schreiber F (2015). The Hd, Hj, and Hz66 flagella variants of Salmonella enterica serovar Typhi modify host responses and cellular interactions. Scientific ReportsDOI:10.1038/srep07947

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Sanger Science

Exploring Salmonella’s deadly sub-Saharan adaptation

Salmonella Typhimurium [Credit: Genome Research Limited]

21 June 2012

Written by Rob Kingsley

I contributed to a literature review article that was published in The Lancet in May 2012 that we hope will produce a wider appreciation of the severity and distinct causes of a disease that is an important cause of death in African adults and children.

Invasive non-typhoidal Salmonella (iNTS) is a blood-borne infection that kills approximately one in four of people in sub-Saharan Africa who catch it. Yet, in the rest of the word, NTS is simply an unpleasant disease: it is a leading cause of acute inflammatory diarrhoea that is self-limiting and tends to be fatal in less than 1 per cent of people.

This difference in the severity of the disease in sub-Saharan Africa when compared with the rest of the world is due, in large part, to synergy with other factors including young age of the person, malnutrition, or coinfection with malaria or HIV. However, our collaborative research with laboratories in Kenya, Malawi and Liverpool using whole-genome sequence analysis has also uncovered an additional complicating factor; the bacterium responsible for the severe disease is distinct from that found in the rest of the world.

In 2009, we announced that a distinct genotype of Salmonella Typhimurium, designated ST313, had emerged as a new pathogenic group in sub-Saharan Africa, and might have adapted to the susceptible population in these regions. While Salmonella Typhimurium genotypes associated with inflammatory diarrhoea have spread globally, the ST313 genotype is specifically associated with susceptible populations in sub-Saharan Africa. The genetic make-up of the pathogen may therefore contribute to the severity and/or epidemiology of this disease. The molecular basis of this association is now the subject of intense interest in laboratories across the world.

I work as part of research consortium that includes the Wellcome Trust Sanger Institute, laboratories in the UK and US, and – most importantly – collaborators from several sub-Sahara African countries, which has contributed significantly to understanding this disease. The collaboration combines local field studies in Malawi, Kenya, Uganda, Democratic Republic of Congo, Mali and Nigeria with molecular and genome sequencing efforts in laboratories around the world.

Despite the significant steps we have taken in understanding this disease in recent years, advocacy for further field studies and the development of effective intervention strategies is lacking. By developing a complete understanding of the epidemiology of this neglected disease, we hope that new avenues will open for development and implementation of vaccine and public health strategies to prevent infections and interrupt transmission.

Rob Kingsley is deputy head of Bacterial genetics within the Microbial Pathogenesis team at the Wellcome Trust Sanger Institute … more

Review article: Feasey et al. Invasive non-typhoidal salmonella disease: an emerging and neglected tropical disease in Africa. Lancet 2012 (Epub ahead of print). doi: http://dx.doi.org/10.1016/S0140-6736(11)61752-2

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