Bacteria: it’s the only culture some people have
Figure 1. A cartoon comparing the number of microbial cells with the number of human cells in and on the human body. The microbial cells can outnumber human cells 10-to-1. Source
We live in a microbial world, surrounded by environments swarming with countless microorganisms -- and our body is no exception. In recent years, the discovery that there are microbes that live in and on us has gained a substantial amount of interest, taking the science world and public attention by storm. We now know the human body contains trillions of microorganisms, a figure so numerous that the microbial cells in this mini-ecosystem -- our human microbiome -- outnumber our own cells 10 to 1! The complex, multi-layered network of our microbiome sets the stage for some very unique and interesting phenomena caused by interactions within its community of microflora. These microorganisms can modulate our brain chemistry by impacting our moods and even influence our health by signalling with our immune systems. Bacteria in particular account for an overwhelming portion of our microbiome and are 2 to 3 times more numerous than our eukaryotic and archaean microbiota from the other two domains of life. Cumulatively, it is believed that there are 100 trillion bacterial cells that live in and on the average human being.
Figure 2. A light microscopy image of Streptococcus pyogenes. Long filaments of the circular bacterium can be seen in orange. Source
One member of our vast microbiome is the spherical bacterium Streptococcus pyogenes. If this critter sounds familiar, that may be because you have heard of how it can cause disease in humans. Indeed, S. pyogenes infections are at the heart of diseases like strep throat, a painful sore throat that accompanies abdominal and digestive symptoms, scarlet fever, a full-body rash that can result in vomiting and light-headedness, and necrotising fasciitis, a flesh-eating infection destroys muscle and fat tissue. In fact, infections caused by S. pyogenes alone are thought to be responsible for over 500,000 deaths in the US each year. In 2005, conservative estimates identified over 740 million people worldwide who became ill from diseases caused by S. pyogenes infections.
Despite this prevalence of infections caused by this species, S. pyogenes mostly colonises the skin and the upper part of the pharynx without causing any infection or discomfort at all. In these environments, S. pyogenes is housed alongside a whole slew of other microbe buddies that collectively comprise our skin microbiota. Thus, S. pyogenes is a type of opportunistic pathogen, a microbe that can coexist happily with our other human microbiota but occasionally spirals out of control to cause burdensome and painful infections. However, in order to cause disease, S. pyogenes needs to first be able to dominate its local environment in which there is already intense competition for resources given the number of microbial “mouths” there are to feed. How is S. pyogenes able to compete with its myriad of neighbours and grow to the extent that it monopolises the energy sources in its environment, spreading rapidly and eventually causing disease?
Researchers at a medical research school in Canada may have found part of the answer.
Started from the bottom now we’re here: using a promoter-trap strategy to identify one of S. pyogenes’ secret weapons
In 2016, graduate students working in the McCormick Lab at the Schulich School of Medicine and Dentistry published a paper in the journal Nature outlining some surprising and clinically-relevant findings that were the culmination of many years of research. The researchers were investigating the possible mechanisms by which S. pyogenes competes with its neighbours to cause disease. More specifically, the team of scientists were searching for proteins or pathways that S. pyogenes may rely on during an infection to overtake the other local microbiota. Where did their search begin? In the S. pyogenes genome.
The researchers developed a novel technique aptly-dubbed the “promoter-trap” strategy that enabled them to screen many fragments of potential promoters that were regulating regions of high gene expression activity during an acute infection of S. pyogenes in the upper pharynx (also known as the nasopharynx) of mice. Promoters are stretches of DNA which occur just upstream of coding sequences and help recruit DNA expression machinery like RNA polymerase to transcribe downstream genes. Promoters themselves do not encode any gene products like an RNA or a protein, but rather help regulate how much or little the genes in its ‘territory’ are read by transcription machinery. Using the promoter-trap technique, the scientists identified a promoter which they named the PspbM promoter which is responsible for regulating a string of genes that collectively form the S. pyogenes bacteriocin (spb) locus. So what exactly are the genes in the spb locus? What are their functions, and how are they related to helping S. pyogenes dominate its niche and out-compete its neighbours? The intriguing aspects of the paper are not limited to the use of the promoter-trap strategy to find PspbM, but also extend to the series of discoveries that came shortly after.
It turns out that the PspbM promoter regulates the expression of an antimicrobial attack system which has some prominent similarities to a group of bacteria-killing molecules known as the class IIb bacteriocins. The antimicrobial attack system encoded by the spb locus was highly expressed during an active S. pyogenes infection in a mouse model but was weakly expressed in a test tube environment, suggesting the locus only becomes highly expressed in an in vivo setting surrounded by a community of other microbes. The S. pyogenes bacteriocin system in particular consists of the two proteins SpbM and SpbN, which are encoded on the spb locus one after the other.
From bacterial brain to bacterial brawn: probing the antimicrobial action of SpbM and SpbN
Bacteriocins ultimately inhibit the growth of closely related strains of bacteria and are thus used widely by many bacteria as a way of eliminating bacterial competitors in a communal environment. For example, Streptococcus pneumoniae, a notorious human pathogen most known for causing infections in respiratory tracts is also known to deploy class IIb bacteriocins in order to cause disease. All class IIb bacteriocins depend on the action of two antimicrobial proteins that work in synergy to kill surrounding bacteria, and these peptides typically target bacterial cell walls. More specifically, the peptides punch many small holes in the cell walls of the target bacteria, forming multiple ion channels that lead to membrane leakage and in most cases cell death. Class IIb bacteriocins cannot effectively act to inhibit competitors’ growth unless both proteins are actively produced in roughly equal concentrations. Likewise, in Figure 5 of their paper (Figure 3 of this post), the researchers from the McCormick Lab show that both and SpbN are necessary for inhibition of other bacteria -- each protein in isolation is not sufficient to sustain that level of antimicrobial activity. The researchers demonstrated this by first covering an entire agar plate with a strain of bacteria called Lactobacillus lactis. Then, the researchers punched small divots or wells into the surface of the agar plate containing L. lactis, into which they spotted purified SpbM and/or SpbN. SpbM and SpbN were first spotted on top of each other, then at increasing distances from one another to determine whether one peptide alone was sufficient to inhibit the growth of L. lactis.
Figure 3. Figure 5 from the research paper by Armstrong et al. (2016) showing that SpbM and SpbN are both necessary for antimicrobial activity against Lactobacillus lactis. SpbM along and SpbN along are not sufficient to sustain any significant levels antimicrobial activity.
In the figure, notice the small halo of darker grey around the well labelled “SpbM + SpbN”. This halo is known as a zone of inhibition and indicates that there is no growth of L. lactis in this zone, which means the peptides are having an antimicrobial effect. However, the zone of inhibition shrinks as the distance between the SpbM and SpbN wells increases. These results show that SpbM alone was only able to weakly inhibit surrounding bacterial growth, given that it still has a very small zone of inhibition surrounding it when it is far away from SpbN, but SpbN on its own had no antimicrobial activity at all evidenced by the absence of a zone of inhibition and the presence of L. lactis around the SpbN well. The researchers repeated this type of experiment many times, swapping out L. lactis for a different bacteria each time. The researchers determined that SpbM and SpbN could inhibit the growth of two other Streptococcus species and Micrococcus luteus in addition to L. lactis.
Immunity to beat out the community: protective genes against SpbM and SpbN are also encoded in the spb locus
With the knowledge that SpbM and SpbN can successfully kill many species of bacteria including other Streptococcus species, the following question may be forming in your mind: isn’t S. pyogenes itself susceptible to the effects of SpbM and SpbN? The answer is yes, although S. pyogenes has another surprise in its toolkit: SpbM and SpbN immunity genes! These genes are also encoded in the spb locus, just after the gene that encodes SpbN. The researchers discovered this locus by copying the chunk of the spb locus hypothesised to contain the immunity genes from S. pyogenes and then transferring the DNA into a species like L. lactis that was previously susceptible to SpbM and SpbN. Strikingly, the researchers found that L. lactis was no longer killed off by SpbM and SpbN once it contained the new DNA from S. pyogenes. Thus, immunity to the antimicrobial peptides was transferred to L. lactis from the immunity genes copied from S. pyogenes. Taken together, these results provide convincing evidence to suggest the presence of a class IIb bacteriocin system in S. pyogenes. Furthermore, these results demonstrate the importance of understanding gene regulation in environments like the diverse microbiota in order to investigate how pathogenic bacteria can adapt to specific niches and eventually overrun them to cause disease in humans and other hosts.
The dawn of sociomicrobiology: the whole is greater than the sum of its parts
The novel S. pyogenes class IIb bacteriocin system discovered by the McCormick lab is but one of the many mechanisms S. pyogenes relies on to thrive in its environment. Once S. pyogenes establishes itself in a specific tissue environment, genes in the bacterial chromosome derived from viruses kick in to help the S. pyogenes further adapt to local conditions. To cause more invasive infections, S. pyogenes can switch to a hypervirulent phenotype that exploits the resources its host has to offer much faster with more devastating consequences for its host. Additionally, old S. pyogenes virulence factors can be up-cycled to form new virulence factors that may enable the bacterium to be a better competitor and/or bacterial pathogen. Each one of these mechanisms can be affected by signalling from neighbouring bacteria, the presence or absence of certain species of bacteria, and the extent of resource competition in any one environment. These ideas belong to the growing field of sociomicrobiology that views one bacterium in the context of the community, a consideration that becomes increasingly relevant with the onset of antibiotic resistance and compromised host immunity.
Research studies like the one by the McCormick lab are part of the school of holistic science that acknowledges the complexity and connectivity in various ecosystems including the human microbiome, and strives to explore the ways in which the ecosystem-level diversity can influence health and disease. An individual cannot be completely isolated from the community in these contexts, and thus the possibility that the community may be able to influence the individual in some way remains plausible. As researchers and caregivers begin to understand the ubiquity of microbes, the impacts of the community on the individual simply cannot be overlooked.
Hi there! My name is Salina Hussain and I am a junior at Mount Holyoke College pursuing a major in biology and a minor in chemistry. I am an aspiring medical doctor and bacteriophage researcher, and the study of viruses and evolutionary biology is my passion! In my free time, I enjoy being outdoors and love reading -- anything from Virgnia Woolf to David Quammen to Roald Dahl! You can reach me at hussa35s@mtholyoke.edu with questions or just if you want to chat to a fellow STEMinist.
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